changes of amylases and carbohydrates in sweetpotatoes

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1995

Changes of Amylases and Carbohydrates inSweetpotatoes During Storage and Their Effects onViscosity of Sweetpotato Puree.Xiangyong LiuLouisiana State University and Agricultural & Mechanical College

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CHANGES OP AMYLASES AND CARBOHYDRATES IN SNEETPOTATOES DURING STORAGE AND THEIR EFPECTS ON VISCOSITY OF

SMEETPOTATO PUREE

A DissertationSubmitted to the Graduate Faculty of the

Louisiana State University and Agricultural and Mechanical College

in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

inThe Department of Horticulture

byXiangyong Liu

B.S., Hunan Agricultural University, 1984 M.S., Hunan Agricultural University, 1987

December 1995

UMI Nunbar: 9613424

UMI Microform 9613424 Copyright 1996, by UMI Com pony. All rights reserved.This microform edition is protected against unauthorized

copying under Title 17, United States Code.

UMI300 North Zeeb Rood Ann Arbor, MI 40103

ACKNOWLKDGEMKNTS

The author wishes to express his sincere appreciation to his major professor, Dr. Paul W. Wilson, Professor, Department of Horticulture, for the encouragement, guidance, patience, and support which he provided throughout the course of this research.

Appreciation is also extended to Dr. David H. Picha, Professor of the Horticultural Department, for his valuable suggestions on sugar analysis by HPLC.

Special thanks are due to Dr. J . Samuel Godber, Dr. Robert M. Grodner, Dr. Don R. Labonte and Dr. David H. Picha for serving as members of the advisory and examining committee toward the completion of this study.

The author wishes to thank Dr, William A. Young, Head, Department of Horticulture, for providing the departmental facilities necessary to conduct this research.

He is sincerely appreciative of Ms. Gloria McClure for tremendous help on sugar analysis by HPLC.

Finally, he wishes to express his deepest love, respect, devotion, and appreciation to his mother Mrs. Zhuping Gao and to his father, the late Mr. Zhengguan Liu, for their love, encouragement, support and understanding during this study.

ii

TABLZ OF CONTENTS

ACKNOWLEDGEMENTS.................................... iiLIST OF T A B L E S ..................................... viLIST OF FIGURES................................... viiiLIST OF ABBREVIATIONS............................... xiA B S T R A C T ............................................ xiiCHAPTER I INTRODUCTION ............................. 1CHAPTER II LITERATURE REVIEW ....................... 5

NUTRITION OF SWEETPOTATO ......................... 5SWEETPOTATO PUREE ............................... 6GENERAL DISCUSSION OF CARBOHYDRATES AND STARCH . 12

Starch Structure ......................... 12Starch Hydrolysis ......................... 14Factors Influencing Starch Digestion . . . 15Gelatinization and Retrogradation ........ 16

CARBOHYDRATES IN SWEETPOTATOES ................. 10Properties of Starch in Sweetpotatoes . . . 18Gelatinization of Sweetpotato Starch . . . 20Enzymatic Hydrolysis of Sweetpotato Starch 21Carbohydrate Changes in Sweetpotatoes During

S t o r a g e ............................. 22Carbohydrate Changes During Heat Treatment 23

GENERAL DISCUSSION OF AMYLASES ................. 24a-Amylase.................................. 24fi-Amylase.................................. 28Temperature Effects on Enzyme Activity and

Thermal Stability ................... 31AMYLASES IN SWEETPOTATOES ..................... 34

Influence of Storage on Sweetpotato AmylaseActivity............................. 35

Thermal Stability of Amylases inSweetpotatoes ....................... 36

DETERMINATION OF a- AND S - A M Y L A S E ............. 37CHAPTER III MATERIALS AND METHODS ............. 43

RAW M A T E R I A L S ............................ 43SAMPLE PREPARATIONS AND ASSAY METHODS ........ 43

Enzyme Assays ............................. 43Protein Determination ..................... 53Dry Matter Determination ................. 54

iii

Puree Preparation......................... 55Determination of Viscosity ............... 55Determination of Alcohol Insoluble Solids

(AIS) ............................... 56Sugar Determination....................... 56Starch Assay ............................. 58

STATISTICAL ANALYSIS ........................... 59EXPERIMENTAL DESIGN AND PROCEDURE ............. 60

Experiment 1. Assay of a- and S-AmylaseActivity............................. 60

Experiment 2. Characterization of SomeProperties of Sweetpotato Amylases . . 65

Experiment 3. Amylases Activity and Carbohydrate changes in Sweetpotatoes during Storage and Their Effects on Puree V i s c o s i t y ..................... 68

CHAPTER IV RESULTS AND DISCUSSION ................. 6 9EXPERIMENT 1. ASSAY OF a- AND S-AMYLASE ACTIVITY 6 9

Linearity of 6PNPG7 or-Amylase AssayP r o c e d u r e ........................... 6 9

Linearity of PNPG5 fi-Amylase AssayP r o c e d u r e ........................... 72

Reproducibility ........................... 76Specificity of Substrates BPNPG7 and PNPG5 79Comparison of Traditional and New Methods for

a-Amylase and S-Amylase Assay........ 83Determination of Km and V m a x ............. 91Temperature effects on a- and fi-amylase

a s s a y ............................... 97Preparation of a-Amylase Reagent ........ 99Preparation of fi-Amylase Reagent ........ 100Calculation of Enzyme Activity .......... 100

EXPERIMENT 2. CHARACTERIZATION OF SOME PROPERTIESOF SWEETPOTATO AMYLASES ................. 102Temperature Effect on Stability of

Amylases............................. 102Comparison of the Stability of Amylases in

Different Cultivars................... 107Dilution Effect on the Stability of

Amylases............................. 110Interaction of Commercial a-Amylase and fi-

Amylase on Starch Hydrolysis ........ 112EXPERIMENT 3. CHANGES OF AMYLASE ACTIVITIES AND

CARBOHYDRATE CONTENTS IN SWEETPOTATOES DURING STORAGE AND THEIR EFFECTS ON VISCOSITY OFSWEETPOTATO PUREE ......................... 119The Effect of Storage on the Activities of a-

and fi-Amylase in Four SweetpotatoCultivars............................. 119

Carbohydrate Changes during Storage and PureeProcessing........................... 127

iv

Effects of Storage on Viscosity ofSweetpotato Puree ................... 145

Suggestions for Improving Consistency ofSweetpotato Puree..................... 154

CHAPTER V SUMMARY AND CONCLUSIONS 157LITERATURE CITED .................................... 163V I T A ................................................ 171

v

LIST OF TABLES

Table 1. Properties of a-amylases from differents o u r c e s .......................................... 2 9

Table 2. Properties of fi-amylases from differents o u r c e s .............................................32

Table 3. Reproducibility of the BPNPG7 and PNPG5 assay for the measurement of sweetpotato a-amylase..and S-amylase.......................... 78

Table 4. Action of fi-amylase on a-amylase assaysubstrate......... BPNPG7.......................... 80

Table 5. Action of a-amylase on fi-amylase assaysubstrates PNPG5 and soluble starch...............81

Table 6. Comparison of sensitivity among threea-amylase assay methods.......................... 89

Table 7. Comparison of sensitivity between twoS-amylase assay methods: PNPG5 and Bernfeldmethods............................................... 90

Table 8. Kinetic properties of sweetpotatoa-amylase for four cultivars for the hydrolysis Of B P N P G 7 ...........................................92

Table 9. Kinetic properties of sweetpotato fi-amylase for four cultivars for'the hydrolysisof PNPG5 ...........................................95

Table 10. Effect of dilution on the stability ofcommercial a-amylase................................ 113

Table 11. Effect of storage time on a-amylaseactivity in four sweetpotato cultivars............. 121

Table 12. Effect of storage time on fi-amylaseactivity in four sweetpotato cultivars ........ 124

Table 13. Comparison of protein content infour sweetpotato cultivars ....................... 126

Table 14. Effect of storage time on reducing sugars(glucose + fructose) in raw sweetpotato roots. . . 130

Table 15. Effect of storage time on maltose producedduring sweetpotato puree processing..................133

Table 16. Effect of storage time on the ratios of maltose produced during processing and AIS in raw roots, the ratios of AIS change during processing and AIS in raw roots. ................ 134

Table 17. Effect of storage time on sucrose contentin raw sweetpotato r o o t s ......................... 13 7

Table 16. Effect of storage time on total sugarcontent in raw sweetpotato roots .................. 13 9

Table 19. Effect of storage time on AIS content inraw sweetpotato roots ........................... 142

Table 20. Change in AIS, Total sugar and drymatter during storage compared to the amounts at harvest ........................................143

Table 21. Effect of storage time on AIS content of sweetpotato puree processed from fourc u l t i v a r s ........................................ 147

Table 22. Effect of storage time on viscosity of sweetpotato puree processed from fourcultivars. ...................................... 149

Table 23. Relationships between viscosity of sweetpotato puree and AIS before processing, AIS after processing, a-amylase activity, and S-amylase activity........................................... 153

Table 24. Alcohol insoluble solids (AIS) contents inraw sweetpotatoes vs viscosity (VIS) of processed puree.............................................. 155

vii

LIST or rioums

Figure 1. Representative fragments and structures ofstarch................................................ 13

Figure 2. Schematic representation of the reaction involved in the measurement of a-amylase using blocked (non-reducing glucosyl group) p-nitrophenyl maltoheptaoside in the presence of excess quantities of a-glucosidase..........................41

Figure 3. Schematic representation of the reaction involved in the measurement of 6-amylase using p-nitrophenyl maltopentaoside (PNPG5) in the presence of excess quantities of a-glucosidase. . 42

Figure 4. Linearity of the BPNPG7 a-amylase assay withcommercial a-amylase concentration.................. 70

Figure 5. Linearity of the BPNPG7 a-amylase assay with concentration of extracted sweetpotato juice................................................. 71

Figure 6. Linearity of the BPNPG7 a-amylase assay with incubation time of a 1/2 dilution of sweetpotato extract.................................. 73

Figure 7. Linearity of the PNPG5 fi-amylase assay withcommercial sweetpotato 6-amylase concentration. . 74

Figure 8. Linearity of the PNPG5 6-amylase assay with concentration of extracted sweetpotato juice................................................. 75

Figure 9. Linearity of the PNPG5 6-amylase assay with incubation time of a 1/8000 dilution of the sweetpotato extract.................................. 77

Figure 10. Relationship between the BPNPG7 and amyloseazure method for measurement of a-amylase........... 84

Figure 11. Relationship between the BPNPG7 and starchazure method for measurement of a-amylase........... 85

Figure 12. Relationship between the BPNPG7 andsoluble starch method (Bernfeld method) for measurement of a-amylase.......................... 86

Figure 13. Relationship between the PNPG5 and Bernfeldmethod for measurement of 6-amylase................. 87

viii

Figure 14. Lineweaver-Burk plots for the hydrolysis of BPNPG7 by sweetpotato a-amylases from four cultivars. . 93

Figure 15. Lineweaver-Burk plots for the hydrolysis of PNPG5 by sweetpotato S-amylases from four cultivars............................................. 94

Figure 16. Effect of temperature on BPNPG7 a-amylaseassay and PNPG5 S-amylase a s s a y .....................98

Figure 17. Stability of a-amylase in sweetpotatoes atdifferent temperatures.........................104

Figure 18. Stability of S-amylase in sweetpotatoes atdifferent temperatures........................ 105

Figure 19. Stability of a-amylase from four differentsweetpotato cultivars at 75°C................. 108

Figure 20. Stability of fi-amylase from four differentsweetpotato cultivars at 75°C................. 109

Figure 21. Effect of dilution of sweetpotato a-amylaseon its heat stability at 75°C................. Ill

Figure 22. Effect of dilution of sweetpotato a-amylaseon its heat stability at 75°C................. 114

Figure 23. Interaction between a-amylase and fi-amylase on sweetpotato starch at high enzymeconcentration...................................... 116

Figure 24. Interaction between a-amylase and S-amylase on sweetpotato starch at normal enzymeconcentration ..................................... 117

Figure 25. a-Amylase activity of four sweetpotato cultivarsat harvest (H), after curing (C), and during fourmonths of storage.................................. 120

Figure 26. S-Amylase activity of four sweetpotato cultivars at harvest (H), after curing (C), and during four months of storage................ 123

Figure 27. Comparison of a- and fi-amylase activity of Jewel cultivar sweetpotatoes harvested in different years at harvest, after curing, and during four or six months of storage . ........ 12 8

Figure 28. Reducing sugar (glucose and fructose) contents in four sweetpotato cultivars at

ix

harvest (H), after curing (C), and duringfour months of storage.............................. 129

Figure 29. The amount of maltose produced (based on fresh weight) in processed sweetpotato puree from four sweetpotato cultivars at harvest (H), after curing (C), and during four months of storage. ..........................................132

Figure 30. Sucrose contents in four sweetpotatocultivars at harvest (H), after curing (C), and during four months of storage. ...................136

Figure 31. Total sugar contents in four sweetpotato cultivars at harvest (H), after curing (C), and during four months of storage. ...................138

Figure 32. Alcohol-insoluble solids (AIS) content of four sweetpotato cultivars at harvest (H), after curing (C), and during four months of storage. . 141

Figure 33. Dry matter contents in four sweetpotato cultivars at harvest (H), after curing (C), and during four months of storage. .............. 144

Figure 34. Alcohol-insoluble solids (AIS) content ofprocessed sweetpotato puree from four sweetpotatocultivars at harvest (H), after curing (C),and during four months of storage. .............14 6

Figure 35. Viscosity of sweetpotato puree processed from four sweetpotato cultivars at harvest (H), after curing (C), and during four months of storage.............................................. 148

x

LIST OF ABBREVIATIONS

ABS AbsorbanceAIS Alcohol insoluble solidsBPNPG7 p-Nitrophenyl maltoheptaosideC.V. Coefficient of variationCPS Centipoise, a unit for viscosity measurementHPLC High performance (or pressure) liquid

chromatographKm Michaelis-Menten constantMr Molecular weightPI Isoelectric pointPNPG5 p-Nitrophenyl maltopentaosideRBB Remazolbrilliant blueRT Room temperature[S] Molar concentration of substrateTCA Trichloroacetic acidU UnitV Initial velocity of the enzyme reactionVmax Maximum velocity (or Substrate turnover number)

ABSTRACT

A critical problem associated with the production of sweetpotato puree is the inconsistency of final product. Two possible factors, amylase activity and carbohydrate content in sweetpotatoes during storage, were investigated. It was found that a- and S-amylase activities do not significantly change during storage, and have no significant effects on viscosity of sweetpotato puree. The inconsistent products in sweetpotato puree processing are mostly due to the change of alcohol insoluble solids (AIS) in sweetpotatoes during storage. The decrease of AIS is partially due to respiration that converts starch into COa and H,0. A new bio-processing method was proposed to improve the consistency in sweetpotato puree products based on the results obtained in this study. In addition, two methods for specific determination of a- and fi-amylase (using blocked p-nitrophenyl maltoheptaoside and p- nitrophenyl maltopentaoside as substrates respectively) were adapted for amylase assays in sweetpotatoes. Both methods have major advantages of simplicity, speed, high sensitivity, and specificity. The thermal stability of native a- and fi- amylase in sweetpotatoes, and the interaction between a- and fi-amylase on starch hydrolysis were also studied. The or- amylase was very heat labile, and lost most activity in just

3 0 seconds of heating at 75°C. S-Amylase had a higher thermal stability. The synergistic hydrolysis of starch could occur when a-amylase is combined with S-amylases, but it is not always true, depending on the concentrations of amylases.

xiii

CHAPTER I INTRODUCTION

The sweetpotato (Ipomoea batatas (Lam.) L.) is a potentially good source of carbohydrates, minerals, dietary fiber, and vitamins suitable for food and industrial uses and is receiving increased consumer attention due to its nutritious character and versatility. The potential for using sweetpotatoes in the development of new products is being realized by food processing industries (Chang-Rupp and Schwartz, 1988b). Processing techniques have been developed to preserve sweetpotatoes for direct use or as an ingredient in other foods. One such technique is to process sweetpotato into puree, one of the most successful commercial sweetpotato products.

A critical problem associated with the production of sweetpotato puree is inconsistent final products because sweetpotato roots undergo internal changes during storage. It was generally believed that the nature of this change was due to the increase of amylase activity in sweetpotatoes. However, puree processing is actually an enzymatic hydrolysis procedure, the consistency of final products could be affected not only by enzyme levels but also by substrate (starch) concentrations. Therefore, to obtain a better understanding of major causes of inconsistency, it is necessary to investigate the effects of both factors (enzyme

1

2levels and carbohydrate contents) on viscosity of puree products.

Sweetpotatoes contain abundant S-amylase and a small amount of a-amylase (Ikemiya and Deobald, 196 6) . These enzymes cause starch breakdown during cooking and thereby strongly influence the textural properties and flavor of the processed products. Hence, amylases have been receiving special attention in sweetpotato processing. Enzyme activity changes during storage among different cultivars have been investigated by many groups (Ikemiya and Deobald, 1966 ; Walter et al. 1975; Morrison et al. 1993). However, results varied greatly among these groups. Also, in the past, determination of E-amylase has been complicated by the presence of a-amylase when using the method described by Bernfeld (1955) , which can be used for both a- and E-amylase determination. As a result, methods for determining E-amylase by measuring formation of reducing sugars, in fact, measures total saccharifying activity of both a- and E-amylase. As to a-amylase determination, the most common method is reported by Rindderknecht et al. (1967) and its modified methods (Hall et al. 1970; Bilderbach, 1973; McCleary, 1900) by using arange of dye-linked chromogenic substrates. These substrates are prepared by reacting starch, starch-fractions, or cross- linked starch with an organic dye (Remazolbrilliant Blue). Previous plant studies using this assay have generally assumed that this substrate is specific for a-amylase and

3that S-amylase is not reactive. However, it was found that the presence of S-amylase had an interfering effect on the release of color from this substrate by or-amylase (Bilderbach, 1973; Doehlert and Duke, 1983) . In addition, cultivars of sweetpotatoes currently grown are different from those used in the early enzyme activity studies. Therefore, the investigation of enzyme activities needs to be performed for the currently grown cultivars using more accurate methods of analysis.

Besides enzyme activity, enzyme stability in vivo at different temperatures is another valuable factor to be considered in food processing. Not only because it provides information concerning proper temperatures to achieve desired reaction rates, but also it gives proper conditions for enzyme preservation and assay. Previously, the stability of sweetpotato amylases was investigated under special circumstances, e.g., after purification, and with added buffer with stabilizer {Hagenimana et al. 1992, Ikemiya and Deobald, 196S). However, most sweetpotato processing is conducted in natural occurring conditions, like baking, without purifying enzyme or adding buffer with stabilizer. Therefore, to provide more realistic information on amylase stabilities to the sweetpotato industry, it is necessary to investigate the properties of the enzymes in crude preparations.

4Recently, more specific methods have been developed for

the determination of a-amylase (McCleary and Sheehan, 1987) and fi-amylase (McCleary and Codd, 1989) in cereals, but no research has been done to investigate activities and stability of native amylases in sweetpotatoes using these methods.

Therefore, the objectives of this study were: (1) toadapt new methods of amylases assay in sweetpotatoes; (2) to investigate a- and fi-amylase activities, carbohydrate contents in sweetpotatoes of currently grown cultivars during storage, and their effects on viscosities of puree; (3) to investigate the stability of amylases in sweetpotatoes in vivo using the new methods.

5

CHAPTER II LITERATURE REVIEW

Sweetpotatoes are presently an important crop in many areas of the world. Statistics derived from the 1990 FAO Production YearBook indicated the world production of sweetpotato increased from 123 million metric tons in 1988 to 131 million metric tons in 1990, ranking seventh among food crops.

Sweetpotatoes are also a major food crop in the United States. Production increased from 4.97 to 5.91 million metric tons during 1988 to 1990 (FAO, 1990).NUTRITION 07 SWEETPOTATO

As a starch root, sweetpotatoes are efficient producers of calories. Among the ten leading food crops, sweetpotatoes rank third in terms of calories produced per square meter (FAO, 1977) .

Sweetpotatoes can make significant nutritional contributions to the diet. while starch root crops are frequently considered to provide only calories to the diet, this is not the case with sweetpotatoes. Based on the nutrient per calorie provided as a percentage of nutrient per calorie required, it was found that sweetpotato roots are an excellent source of most nutrients, providing at least 90% of the RDA requirement for all except protein and niacin (Food and Nutrition Board, 1980; Watt and Merrill, 1975).

6SWEETPOTATO PUREE

The sweetpotato processing industry has developed many processing techniques to preserve sweetpotato for direct use or as an ingredient in other foods as a result of the following important considerations: (1) the convenience ofprocessed product contributed to its desirability; (2) the off grade product was priced at a level such that the processed product was economically competitive with comparable commodities; (3) the processed sweetpotato products are subjected to much less loses than that of the raw product during storage and marketing; (4) the processed sweetpotato products cost less during storage because of lower usage of energy and space; (5) the nutrition ofprocessed sweetpotato products can be further improved by adding other ingredients, e.g. soy protein. Since the development of processed products from sweetpotatoes represents perhaps one of the most important keys to expanded utilization of the crop, studies have thus been initiated to explore methods of increasing the usability of sweetpotatoes through processing, e.g. canning, freezing, and dehydration, etc. One such method is to process sweetpotatoes into puree, one of the most successful commercial sweetpotato products.

Puree is prepared by the conventional steps of peeling, trimming, cooking in water or steam, and pulping or screening to break up large particles and to remove fiber or other undesirable materials.

7One of the major advantages of the use of a puree for

processed products is that there are no size or shape criteria for the roots. A high quality puree can be produced from virtually any size or shape of storage roots, so the entire crop can be utilized. Since an aseptically processed puree can be stored at ambient temperatures in bulk containers, there is a minimal storage energy requirement after initial processing. Other advantages include a year- round supply and a substantially reduced product volume for storage (Kays, 1985).

The puree can then be preserved directly by canning or freezing or further processing by dehydration, to remove a great deal of bulk. Sweetpotatoes can be pureed to produce a product that may be used for baby food, pie filling, reconstituted potatoes, and frozen sweetpotato patties. A puree is also utilized in the production of sweetpotato flakes and numerous other products. However, the use of sweetpotato puree as an infant food represents the most successful processed sweetpotato product.

A critical problem associated with the production of sweetpotato puree is inconsistent final product since sweetpotato roots undergo internal changes during storage. The nature of these changes is due in part to the increase in activity of amylolytic enzymes (Walter et al. 1975). These enzymes cause starch breakdown during cooking and increased sweetness thereby strongly influencing the textural

8properties and flavor of the processed products. During enzymatic hydrolysis, the amount of final products produced is not only governed by enzyme activity but also substrate concentration. It was reported that starch content in sweetpotato decreased and sugar level increased during storage {Picha, 1987). Therefore, carbohydrate changes during storage could also be an important factor. Another problem is high starch content in freshly harvested uncured roots. If puree is made from these roots, it tends to retrograde after cooling or during storage. Also, the flakes made from these purees are rather porous, with a low bulk density, and are generally considered inferior in quality.

The use of native enzymes from sweetpotatoes or microbial amylases to solve these problems is the best choice to date. Compared to purely chemical reaction mechanisms, enzymes have many advantages. These include: the speed ofreaction; the fact that the reaction takes place under mild conditions; and, most importantly, enzymes are highly specific. The mild conditions reduce energy costs and high specificity minimizes the need for downstream processing. Recently, the American consumer has shown an interest in less-processed, fresher-tasting retail food products. At the same time, traditional food additives, which have been responsible for significant advances in taste, shelf life, and other quality attributes, have been become less acceptable to the consumer. Enzyme treatment offers a way to

9achieve very specific changes in whole food or food ingredients, where the enzyme is removed or inactivated, the altered food require no specific label reference to enzyme use. The increase in the availability and purity of commercial enzymes also makes enzyme treatment more attractive. In addition, the sweetpotato contains abundant E- amylase throughout the root (Hagenimana et al. 1992), which can be the cheapest enzyme source for sweetpotato processing.

Various procedures have been developed for improving the quality of puree. Deobald et al. (1962) found that under some conditions, holding the cured roots at 74°C for 30 min prior to peeling resulted in improved quality. It was suggested that when raw whole roots were cooked or preheated, a barrier was retained, which could prevent rapid action of amylases on the sweetpotato starch. Therefore, enzyme activity could be controlled before over-conversion of starch occurred. Hoover (1966) produced acceptable flakes from uncured sweetpotatoes by adding an amylolytic enzyme (Rhozymes) after cooking 25 min at 100°C in a steam cooker and pulping the roots. The amount of starch hydrolysis could be controlled by controlling the time the enzyme was allowed to act before the enzymes were inactivated by heating the puree in a tubular heat exchanger to 88°C or above and by regulating the amount of enzyme treated puree added back to the nontreated puree before drying.

10Spadaro et al. (1967) reported on modifications of an

earlier procedure (Spadaro and Patton, 1961) which had been developed for the production of flakes from the cured roots of the cultivar 'Goldrush.' They found that additions of amylase and/or sucrose after cooking and pureeing would result in flakes which, on rehydration, were acceptable. The specific combinations depended on the cultivar used and on the length of storage after curing. Bertoniere et al. (1966)found that curing and storing affected the optimal amount of amylase required for acceptable flakes.

A second procedure for controlling the quality of the processed product was known as " enzyme activation" as described by Hoover (1967). The process includes peeling, comminuting to 0 .7 mm, raising the temperature rapidly to 70- 85°C, holding for specific time before heating to 103°C to inactivate enzymes, then drying on a drum dryer. Holding temperatures of 79-85°C were found to be optimum for the activity of the native amylase (Hoover and Harmon, 1967). Lower or higher temperatures resulted in reduced conversion of starches to maltose. Approximately 90% of the conversion occurred within the first 10 minutes although the quality of the flakes continued to increase with holding times up to 60 min, due possibly to changes in viscosity of the remaining starch(Walter et al. 1976).

Deobald et al. (1968, 1969) found that differences in a- amylase activity of the raw roots measured by a procedure

11developed by Ikemiya and Deobald (1966) could be related to processing characteristics and quality of the dehydrated flakes produced. Amylase activity could be enhanced in freshly harvested roots by additions of calcium ions and by increasing levels of amylase. These could be reduced by longer preheating at higher temperatures and reducing the conversion time.

Szyperski et al. (1986) developed a process where puree, previously processed by steam injection to inactive the native amylases, was treated with exogenous a-amylase. The hydrolyzed puree was then blended with untreated portions to produce a puree with consistent viscosity.

In order to use viscosity as a controlling parameter, the flow properties of sweetpotato puree, as influenced by starch hydrolysis, were investigated. Rao et al. (1975a)studied the flow behavior of sweetpotato puree and its relationship to mouthfeel quality. This work indicated that yield stress was correlated significantly to sensory panel scores. Rao et al. (1975b) demonstrated that apparentviscosity could be used as a means of classifying sweetpotatoes to predict the degree of moist mouthfeel. Rao and Graham (1982) combined rheological, chemical, and sensory data to characterize sweetpotato flakes. They found that viscosity correlated significantly with both chemical properties and sensory texture panel notes.

12GENERAL DISCUSSION OF CARBOHYDRATES AND STARCH

Carbohydrates represent a generic class of substances that display wide biochemical diversity and unparalleled abundance throughout the biosphere. Carbohydrates contribute to the fundamental structures of plants, animals, and microorganisms in addition to supplying a source of biochemical energy that supports life processes and reproduction. Among the most important types of carbohydrates are starch, sugars, dextrins, celluloses, hemicelluloses, pectins, and certain gums. Chemically, carbohydrates contain only the elements carbon, hydrogen, and oxygen.Starch Structure

One of the simplest carbohydrates is the six-carbon sugar glucose. Two glucose units may be linked together with the splitting out of a molecule of water. The result is the formation of a molecule of a disaccharide, maltose. A larger number of glucose units may be linked together in polymer fashion to form polysaccharides. One such polysaccharide is amylose, an important component of plant starches (Figure 1). The molecular structure of amylose is linear and its glucose units are repet itively linked at a-1,4 positions. Molecular weights for amylose range from a few thousand to 150,000. Amylopectin also has a linear structure identical to amylose, but in addition to the a-1,4 glycosidic linkage, periodic glucose residues show a-1,6 glycosidic bonding to linear chain (Figure 1) forming branched structure.

13

C H P H C H p H C H p H---Q t-- D O

OH O OH O OH O

OH OH OH

CHpH CHpHO O

OH O OH -

_--- QOH HO

CHpH CH, C H p H

— o — o o

OH O OH O OH O

OH OH OH

.......... . ■ ' vr . I' - * - .. .

Figure 1. Representative fragments and structures of starch.

14Starch Hydrolysis

Starch molecules may be fragmented by acid hydrolysis conducted at pH 2.0 or by enzymatic hydrolysis. However, enzymatic hydrolysis has shown increased importance in industrial carbohydrate processing because of many advantages over traditional acid hydrolysis, e.g. mild processing condition, substrate specific, environmentally sound, more predictable hydrolytic behavior, etc.

The a-amylases are classified as endoglucosidases because they randomly attack amylose at a-1,4 linkages to produce simpler sugars such as maltose and glucose, as well as short polysaccharide chains called dextrins.

Other enzymes known as fi-amylases are present in plants (e.g., barley malt, sweetpotatoes, wheat, soybeans), but these enzymes exhibit exoglycosidic activity. That is, they attack the nonreducing end of amylose to produce successive units of maltose.

Amylopectin is susceptible to enzymatic hydrolysis by a- and S-amylases, but its molecular branching cannot be hydrolyzed by these enzymes. Moreover, the singular use of 6- amylase on amylopectin results in hydrolysis of the linear molecular portions (from the nonreducing end) to within two or three glucose residues of the a-1,6 linkage. Since dextrins of this sort impede further enzymatic hydrolysis, these branching structures are called S-limit dextrins.

15S-Limit dextrins can be enzymatically hydrolyzed at the

cr-1,6-position, however, through the action of a-1,6- glucosidase, and this will permit further polysaccharide hydrolysis by G-amylase. Enzymes that cleave these a-1,6 linkages are generally called branching enzymes.

Another important enzyme called glucoamylase consecutively hydrolyzes a-1,4 linkage found in starch and also hydrolyzes and liberates glucose residues held in of-1,6 linkages at a somewhat lower rate. The enzyme is classified as an exoenzyme and initiates its hydrolysis activity at the nonreducing end.Factors Influencing Starch Digestion

The digest ion of starch by a - amylase has been the subject of many investigations in recent years due to its analytical and possible nutritional significance in the dietary fiber concept. In general, these studies have revealed that susceptibility of starch to amylolytic enzymes, depends not only on the source of starch and enzyme, but also on the processing and storage conditions to which starch is subjected. For example, cooking greatly improves the digestibility of poorly-digestible starch, presumably due to granular disorganization and changes in crystallinity of starch materials. Furthermore, incompletely gelatinized products of whole wheat exhibit reduced rates of cr-amylolysis and thus elicit low glycemic responses. Other factors known to affect the kinetics and extent of a-amylolysis of starch

16include amyloee-amylopectin ratio and retrogradation of starch molecules. Furthermore, since enzymatic hydrolysis of starch-containing foods is a heterogeneous reaction, available surface area is an important parameter. The latter is related to granule and particle sizes (coarse vs. finely- ground materials), macrostructure (compact vs. porous) and the presence of other constituents (e.g. cell walls, proteins) which may act as physical barriers (entrapment of starch) and thereby restrict enzyme accessibility to starch. Depending on the severity of conditions, thermal processing affects the physical state and form of starch and, therefore, can influence starch availability and glycemic response; this was shown for extruded and drum-dried wheat flour products (Seneviratne and Biliaderis, 1991).Oelatinization and Ratrogradation

When starch is suspended in water and heated to a critical temperature, the crystallites within the granules lose their birefringent properties, swell and eventually lose their absolute structural identity. Moreover, as a typical aqueous suspension of starch granules increases in viscosity, the opacity of granules is lost, and a paste is ultimately formed. This overall process is called gelatinization. Heat gelat inizat ion does not occur all at once at a specific temperature, but over a range of about 10°C. This temperature range varies with starches from different sources.

17Those starches with high amylose content are more

resistant to gelatinization because amylose is a highly associated linear molecule (Lee, 1983) . The aqueous solution of amylose leached from native granules during heating may readily form an insoluble precipitate as the solution cools. This precipitate results because the linear molecules tend to line up parallel to one another, which causes association through hydrogen bonding, thus decreasing the affinity of water. The aggregate size increases, and a precipitate is formed. The precipitate is called retrograded starch, and the phenomenon is known as retrogradation (Lee, 1983) . Rapid cooling results in the establishment of intermolecular association with a large amount of solvent entrapped within the gelled molecular network. Gradual cooling results in formation of a two-phase system (solid-starch/liquid- solvent). Linear polysaccharides having about 2 000 glucose unit (e.g., potato starch) and dextrins of less than 30 units usually display poor retrogradation, while starch molecules in the range of 300 to S00 glucose units (e.g., corn starch) show significant binding interactions and retrogradation. Concentrated solutions of amylopectin will undergo macromolecular aggregation, especially if they experience repeated freeze-thaw transition, but the typical a-1,6 branching of these starch molecules diminishes their molecular aggregation when compared with amylose (Zapsalis and Beck, 1985) .

18CARBOHYDRATES IN SWEETPOTATOES

The dry matter portion of sweetpotatoes is mostly composed of carbohydrates. These carbohydrates exist primarily in the form of starches, sugars, and celluloses. Sucrose is the major sugar in unprocessed sweetpotatoes and glucose and fructose are the main reducing sugars (Picha, 1987) .Properties of Starch in Sweetpotatoes

Madamba et al. (1975) reported amylose contents of sweetpotato starches to be from 29.4 to 32.2% with small but statistically significant differences among the six cultivars ('Daja', 'Georgia Red', 'Sweetpotato 45', 'Centennial', 'Jewel' and 'BNAS') under study. Uehara (1983) found an amylose content of 21.6% in sweetpotato starch. It was also determined that treatment of starch with 7 M urea for 1 hr at 30°C resulted in solubilization of 93.6% of the amylose fraction while 99.8% of the amylopectin remained in the insoluble fraction. Takeda et al. (1986) reported that the amylose content of sweetpotato starch in 'Koganesengan' and 'Minamiyutaka' cultivar ranged from 17.2-19.0%. Watanabe et al . (1982) reported that the amylose content of sweetpotato starch was 20.9%. Doremus et al.(1951) found a range of amylose content of 17.5 to 21.7% in twenty-two cultivars, which included 'Triumph', 'Nancy Hall', 'Pelican Processor', 'Porto Rico', 'White Star', 'Australian Canner', 'Ranger', 'Nancy Cold', 'Dessert', 'Queen Mary', etc. Others

19{Takahashi, 1966, Bertoniere et al. 1966) have reported about 18% amylose from individual cultivars.

Sweetpotato starch grains are of variable shapes {oval, round, faceted-round, and polygonal) and are generally nonaggregated (Madamfca et al. 1975, Thurber et al. 1963).Granule diameters range from 4 to 43 microns, depending on the cultivar, with cultivar mean granule size ranging from 12.3 to 21.5 microns. Barham et al. (1944) found starchgrains in the five sweetpotato cultivars studied ranged from 2 to 26 microns in diameter with cultivar means ranging from 8.49 to 9.50 microns. Curing the roots results in starchgrains averaging about 1 micron smaller than starch grainsextracted from freshly harvested roots. Barham and Wagoner (1946) reported that the easily extracted starch grains were generally smaller than the more difficult to extract grains. Madamba et al. (1975) found a large and statistically significant range of phosphorus content in sweetpotato starch from 9 to 22 mg/IOC g. The intrinsic viscosities of starches from six cultivars ('Daja', 'Georgia Red', 'Sweetpotato 45', 'Centennial', 'Jewel' and 'ENAS') were found to be 129 to 155 ml/g. This indicates that sweetpotato starches are net as highly polymerized as potato starch and are more like cereal starches. They found no relation between intrinsic viscosity and particle size. Rasper (1969) found a maximum viscosity of sweetpotato starch of 590 Brabender units, slightly less

20viscous than gelatinized cassava starch but more viscous than corn starch.Gelatinization of Sweetpotato Starch

Average gelatinization temperature for starch from six cultivars of sweetpotatoes ('Daja', 'Georgia Red','Sweetpotato 45' , 'Centennial', 'Jewel' and 'BNAS') was found to range from 63.6 to 70.7°c by Madamba et al. (1975) . Asignificant positive correlation was found between average gelatinization temperature and amylose content of the starch. Gelatinization occurred over a range of 12 to 17CC of temperature change. Barham et al,{1944) found starch from five cultivars had average gelatinization temperatures from 69.0 to 75.5 °C and that the average gelatinization temperature was reduced after curing the roots. Rasper(1969) reported that sweetpotato starch began to gelatinize at 77°C and continued to increase in viscosity until a temperature of 85°C was attained. Sweetpotato starch exhibits a single stage swelling pattern. Swelling potential of six cultivars was found to be in the range of 18 to 26 g of water absorbed per gram of residual starch (Madamba et al. 1975). The swelling power curves followed the same pattern as the solubility curves, indicating a direct relation of these functions. Kohyama and Nishinari (1991) found gelatinization peak temperature increased with increasing sugar concentration contained in sweetpotatoes. The effect was larger in the order sucrose, glucose, fructose. Each of these sugars

21prevented retrogradation of sweetpotato starch paste, and the order of effectiveness was the same as the order of increasing gelatinization temperature.Enzymatic Hydrolysis of Sweetpotato Starch

The susceptibility of sweetpotato starch to a-amylase after one day incubation at gelatinization temperature was found to range from 35.7 to 65.5% weight loss among the six cultivars tested (Madamba et al. 1975). A highly significant negative correlation (-0.91) between amylosis loss and average size of starch grains was reported. Rasper (1969) also noted among "root" starches smaller starch grains were more susceptible to enzymatic degradation.

The action pattern of Bacillus subtilis a-amylase on 'Jewel' variety sweetpotato starch, amylose and amylopectin was reported by Chang-Rupp and Schwartz (1988a) . A wide range of products from large molecular weight polysaccharides to specific low molecular weight compounds were observed during of-amylolysis of sweetpotato starch at 40°C. The initial hydrolysis of amylose alone formed high molecular weight components followed by further hydrolysis to oligosaccharide. This hydrolysis pattern for amylose in starch was concealed by hydrolytic fragments formed from amylolysis of the greater quantity of amylopectin. The amylopectin portion of sweetpotato starch was degraded by a-amylase to more specific polysaccharide fragments. Changes in molecular size of hydrolytic fragments during amylolysis was demonstrated by

22the cluster model: enzyme attack occurs most readily in the more open accessible region where clusters are less dense. It was suggested a nonrandom mode of action on amylopectin and accounts for the pattern of hydrolysis observed in sweetpotato starch.

Baba and Kainuma (1987) studied the hydrolyses of sweetpotato starch by 15-amylase. They found that the hydrolysis of amylopectin components was near-linearly proportional to increasing treatment time in the range between 0 and 40% 6-amylolysis. However, very little degradation of amylose components was shown to take place at the beginning of hydrolysis. An increased rate of amylose hydrolysis was found after 40% of starch was hydrolyzed by 15- amylase. The results demonstrate that most of the amylose component remains even after the greater part of the starch is hydrolyzed by E-amylase. It was suggested that the abundant numbers of nonreducing ends of amylopectin probably causes its faster hydrolysis than that of amylose in the initial stage of S-amylolysis.Carbohydrate Changea in Sweetpotatoea During Storage

For both 'Jewel' and 'Centennial' cultivars, as length of storage increased, starch content decreased and sugar level increased (Walter and Hoover, 1984; Picha, 1987) . The greatest increase in sugar concentration generally occurred during the curing period. In addition, fructose and glucose content increased more rapidly in 'Jewel' than in

23'Centennial' during storage. Sucrose levels increased during storage in 'Centennial' more than in 'Jewel' until 16-wk. The total sugar content, however, was similar for both varieties. No maltose was detected in raw roots.Carbohydrate Changes During Keat Treatment

Carbohydrate changes occurring in sweetpotatoes during enzymatic conversion of starch have been studied by several groups. Core (1923), Sistrunk et al. (1954), and Lambous(1958) have reported an increase in maltose during heat treatment as in baking and cooking. Hoover and Harmon (1967) found that maltose is the only sugar produced and its production is 90% complete after 10 min at 75°C (The amount of maltose produced at the end of two hours was defined as 100% conversion). For both 'Centennial' and 'Jewel', the amount of maltose produced during baking was very close (Walter et al. 1975), with 14.6% (on fresh weight base) for 'Centennial' and 14.2% for 'Jewel' at harvest. Values decreased to 10.8% for 'Centennial' and 9.9% for 'Jewel' after seventy one days of storage. Cooking caused a significant decrease in starch content and considerable maltose formation, which reflected heat-mediated enzymatic conversion of the starch. Walter and Hoover (1984) reported that the maltose content and starch remaining after cooking decreased with the length of storage of the raw roots prior to cooking. This may be due to the decrease in total starch content during storage. For 'Jewel' the starch conversion

24rates (amount of starch change during cooking/amount of starch in raw sample) decreased from 38% at harvest to 26% after roots had been stored for 6 months. 'Centennial' starch conversion rates were relatively stable at between 33-40%. GENERAL DISCUSSION OF AMYLASES

Alpha- and beta-amylases are two of most important enzymes in the food industry. Like other enzymes, they are proteins with a special ability to catalyze specific chemical reactions. Regardless of source and the purity of an amylase preparation, amylase action is characterized by the simultaneous changes of the following properties of the starch: {a) decrease in viscosity of aqueous solution, (b)increase of reducing power, (c) change in iodine-color reaction, (d) change in optical rotatory power (Fuwa, 1954). a-Amylase

a-amylase [a(l-»4) glucan 4-glucanohydrolase, EC 3.2.1.1. ] occurs widely in the animal, plant, and microbial kingdoms with the important industrial sources being cereal, Aspergillus species and Bacillus species. All a-amylases are calcium metallo-enzymes having between one to ten atoms of calcium per molecule of enzyme; the binding strength of the protein for calcium is dependent on the enzyme source. Enzyme activity and conformational stability, which results in stability towards extremes of pH, temperature, and exposure to certain proteases, require one calcium atom per molecule {White and Kennedy, 1988). Molecular weights (Mw) of a-

25amylase vary considerably from 22,500 D for Bacillus lichniformis to 68,000 D for Bacillus acidocaldarius although the majority of a-amylase have molecular weights within the range 47,000-52,000 {Fogarty and Kelly, 1990). The pH for optimum activity in a-amylase is normally between pH 4 . 8 and 6.5. The pH optimum for plant and microbial a-amylase is generally lower than for enzymes from animal origin. Temperature-activity optima are also dependent on enzyme origin with some enzymes being sufficiently stable to allow starch hydrolysis at 70-80°C.

In one important commercial use, a-amylase is employed to convert starch to dextrin, with sufficient hydrolysis occurring to make the products soluble and not susceptible to gelling upon cooling. Historically, the production of dextrin by a-amylase hydrolysis occurred in two steps, cooking of a starch slurry at a temperature of 100°C or higher to swell and gelatinize the starch granules, followed by prolonged enzymatic hydrolysis at temperatures of 80 to 95°C. More recently, upon discovery of more thermostable a-amylase varieties, the process has been streamlined, with starch slurry containing a-amylase being cooked and liquefied at slightly above 100°C by continuously passing the mixture through a jet cooker to which steam is added (Reilly, 1980) . Action of a-amvlase

a-amylase hydrolyzes interior a- (1,4) linkages by an endo-acting mechanism to give random hydrolysis at any of the

26Of-(1,4)-linkages within the amylose and amylopectin molecules. This causes rapid reduction in the molecular size of starch and hence the viscosity of starch solutions. Hydrolysis of amylose will, if allowed to continue for sufficient time, produce maltose and maltotriose. Maltotriose is a poor substrate for a-amylase, the ultimate conversion to maltose and glucose (the second stage of hydrolysis) is slow. Because the enzyme is unable to cleave the a(l-*6) linkages of amylopectin, an a-limit dextrin results plus maltose upon prolonged hydrolysis (Thoma et al. 1971). Each of the a-limit dextrins contains at least one (1,6)-linkage. As with amylose hydrolysis, the second stage of hydrolysis of products from the initial amylopectin hydrolysis is slow but does result in some hydrolysis of specific (1,4)-linkages adjacent to the {1,6)-linkages of the a-limit dextrins. Different a-amylases produce different a-limit dextrins from the same substrate and the study of the action pattern of many enzymes has led to the conclusion that, after the initial random attack on a long polymer chain, the enzyme releases only one of the resulting fragments. The retained fragment may be repeatedly hydrolyzed near one end with the series of oligosaccharides (Thoma, 1976).Properties.of microbial q-amylaseA. Bacterial a-amylase

The a-amylase developed for industrial use from B. amyloliquefacienB was initially called B. mesentericus, then

27fl. subtilis, and now B. amyloliquefaciens. The a-amylase is quite heat-stable and may be used in starch hydrolysis up to 90°C. In 1983, it was found that a-amylase produced by B. licheniformia CUMC 3 05 had maximal activity at 90°C and pH9.0, and in the presence of substrate (starch) was fully stable at lOO^C for four hours (Krishman and Chandra, 1983) .

The Bacillus amylases are metalloenzymes. The B. amyloliquefaciens amylase consists of four subunits bound together by one zinc atom. The subunits are separable, and enzyme molecules with multiple subunits may be formed, but the four-subunit structure has the highest activity (White and Kennedy, 1988) .

Calcium ion stabilizes the enzymes and is customarily added to the reaction liquids. Enhancement of the activity by Ca1* is based on the ability of the ion to interact with the negatively-charged site of the amino acid residues and, thus, to bring about stabilization of the enzyme conformation (Belitz and Grosch, 1987). Amylase from B. licheniformis is less dependent upon Cas* stabilization than the enzyme from B. amyloliquefaciens.

B . Fungal a-amylasea-Amylase is the main component of the old enzyme

preparation "Taka-diastase" prepared from Aspergillus oryzae. The enzyme is called Taka a-amylase by some authors. The molecule is different from the Bacillus amylase in a number of ways: no subunits have been detected; it contains eight

28half-cystine groups and one SH group; it is a glycoprotein with 8 moles mannose, 1 mole xylose, and 2 moles hexosamine. Ten calcium ions are associated with the molecule, nine of which may be removed by dialysis. The molecular weight is51,000. A. oryzae amylase has pH optimum at 4.8-5.8, and it is less heat-stable than B. amyloliquefaciens amylase.

o-Amylase is also produced by A. niger. The properties of this enzyme are similar to those of the A. oryzae enzymes, but some A. niger strains produce an additional acid amylase that is fairly stable down to pH 2 and somewhat more heat- stable. Despite the obvious practical advantages of this enzyme, it has found only limited application, probably because of low yield and, consequently, high price.

Properties of some a-amylases from different sources are summarized in Table 1. fi-Amylase

fi-Amylase [or(l-*4) glucan maltohydrolase, EC 3.2.1.2] occurs widely in many type of plants and, until recently, plants such as barley, wheat, sweetpotato, and soybean have been the commercial sources of the enzyme. All plant S- amylases are sulphydryl enzymes and have no apparent requirements for metal ions for activity or stability. The pH for optimum activity of the plant enzymes is between pH 5.0 and 6.0 while the microbial enzymes have pH optima nearer to neutrality in the range pH 6.0-7.0. Most of S-amylase have temperatures for optimum activity of about 4 5°C.

29Table 1. Properties of a-amylases from different sources

Enzyme source Optimum pH Optimum Temp.(°C)

Bacillus amyloliquefaciens 5 . 5-7.0 90Aspergillus oryzae 5-7 50-55Badllus megaterium 5.5 75B a d 1 lus 1 icheniformis 9.0 90Bacillus subtilis 6-7Lactobacillus cellobiosus D-3 9 7 . 3 50Thermoactinomyces sp. 7.0 80Schwanniomyces alluvius 6 . 3 40Fi1obasidium capsuligenum 5 . 6 50Aspergillus kawachii I 4-5 70Aspergill us kawachii II 5 . 0 70Aspergillus awamori 4 . 8-5.0 50Sweet potato 6 . 0 70-75

Source: Fogarty and Kelly(1990)

30Action of 6-amylase

Beta-amylase cleaves alternate a-(l-*4) D-glucosidic linkages in starch components in a stepwise fashion from the nonreducing end, resulting in the production of 6-maltose. The action of the enzyme stops in the region of ar-(l-*6) D- glucosidic linkages. Complete degradation of amylose and linear dextrin chains containing an even number of D-glucose residues results in production of maltose, whereas those with an odd number result in the production of maltose and a single D-glucose residue. Degradation of branched starch molecules, such as amylopectin, results in the formation of maltose and a 6-limit dextrin (Kruger and Lineback, 1987). Properties of some microbial. S-amylasesA. Bacillus polymyxa 6-amylase

The amylase system of Bacillus polymyxa was studied by Rose (194 8) and Robyt and French (1964). It produced maltose in a yield of 92-94% from starch and was thought to posses an endo-mechanism of substrate attack and have the ability to break or bypass a-1,6-linkage in amylopectin or starch (Robyt and French, 1964). Using high resolution techniques, the enzyme system was resolved into two components - a 6-amylase and a debranching enzyme (Fogarty and Griffin, 1975). The establishment and identification of the presence of two enzymes explained the very high yields of maltose obtained with the system. It also established for the first time the presence of 6-amylase in microorganisms. The 6-amylase was

31similar in many of its properties to the corresponding plant enzymes (Fogarty and Griffin, 1975). It was inhibited by Schardinger cyclodextrins, p-chloromercuribenzoate (p-CMP) and n-bromosuccinmide. Inhibition by p-CMB could be reversed in the presence of cysteine and mercaptoethanol. The optimum pH of 6-8 is higher than that detected in similar plant enzymes. The enzyme has a molecular weight (Mr) of 59,000 (Fogarty and Kelly, 1990) .B. Other microbial 6-amylases

Following its isolation and characterization in culture filtrates of B . polymyxa, 6-amylase has been detected in a number of bacteria and principally in the genus Bacillus (Table 2) . Recent work has dealt with other 6-amylase- producing bacteria. Bacillus cereus strain BQ10-SI was isolated following UV-irradiation of wild-type BQ10 (Shinke et al. 1979) and secreted ten times more 6-amylase than its parent.Temperature Effects on Enzyme Activity and Thermal Stability

The enzymatic process is influenced the most by the temperature to which food is exposed during processing and storage. As temperature rises, the rates of chemical reactions increase, but high temperatures cause denaturation so that enzymes lose activity. Combination of these effects leads to a bell-shaped curve for the variation of activity with temperature. The "temperature optimum" can be used on iy conditionally to characterize the enzyme since its position

Table 2. Properties of S-amylases from different sources

ORGANISM PH OPTIMUM TEMP OPTIMUM(°C) PI MRBacillus cereus 7.0 40 8.3 62 000The mosulphurogenesB-12 -1 5.5 75 5.1 210 000Bacillus sp. IMD 198 6.8 55 --- 58 000Bacillus circulans 7.0-7.5 60 4.5 60 000Bacillus sp. IMD 273 6.5-7.0 50 5.5 57 000Bacillus megateriurn G-2 7.0 60 --- 60 000Sweet Potato 5.0-6.0 50 4.8 152,000

Fogarty and Kelly (1990)

wto

33depends on assay conditions. The longer the enzyme is exposed to higher temperatures, the greater amount of enzyme being inactivated or denatured; hence, the lower is the observed "temperature optimum". Therefore, the term "temperature optimum" is more an operational parameter than a reliable characteristic of enzyme.

The thermal stability of enzyme is quite variable. For example, it was found that a-amylase produced by B. licheniformis CUMC 305 had maximal activity at 90°C and pH9.0, and in the presence of substrate (starch) was fully stable at 100°C for four hours (Krishman and Chandra, 1983) . But a-amylase from potato is so heat labile that several workers have concluded that it is not feasible to measure its activity (Rose and Davis, 1987; Morrell and ap Rees, 1986). All the factors that may cause the denaturation of protein can also cause enzyme inactivation. In the case of enzyme the consequences are easily observed since a slight conformation change at the active site can result in total loss of activity (Belitz and Grosch, 1987).

Thermal inactivation of enzymes is believed to occur in two stages. The first step, which is reversible, is partial unfolding of the protein molecule. The second step, which is irreversible, depends on the enzyme and is either a covalent or a non-covalent reaction. The non-covalent changes are either incorrect folding or aggregation of unfolded molecules (Righelato and Rodgers, 1985).

34AMYLASES IN SWEETPOTATOES

The amylolytic enzyme system of sweetpotatoes has been shown to consist mainly of S-amylase and a smaller amount of a-amylase (Ikemiya and Deobald, 1966) . The presence of an active diastase in sweetpotato was demonstrated as early as in 1920 by Gore. He reported that slow cooking of the roots through a range of 60-100°C converted large amounts of starch into soluble carbohydrates. Giri (1934) reported that sweetpotato amylases were similar to malt S-amylase. The sweetpotato S-amylase was first purified to a crystalline state by Balls et al.(1948). Takeda (1969) reported an improved method for crystallization of S-amylase without detectable a-amylase activity. Recently, Hagenimana et al . (1992) applied more advanced technology to purify sweetpotato a- and S-amylases with 662-fold and 24-fold increases, respectively. It was found that purified sweetpotato a- amylase had the highest activity at 70°C . Purifiedsweetpotato S-amylase was most active at 50°C. Both amylases were unable to hydrolyze native sweetpotato starch granules.

Ikemiya and Deobald(1966) isolated sweetpotato a-amylase that had characteristics of high temperature optimum (70- 75°C), heat stability, and low activity at ordinary temperatures. It was also reported that a-amylase is distributed uniformly throughout the inner tissue, with the outer layer and skin being low in that enzyme. However, different results were obtained by Hagenimana et al. (1992) .

35The highest level of a-amylase activity was found in the outer tissue of the root; this was found for all uncured sweetpotato cultivars. The a-amylase activity in the outer tissue was six times that of the inner tissue in 'Regal', three times in 'Jewel' and "Porto Rico, and four times in 'White Delight'. 'Porto Rico' showed the highest a-amylase activity and 'Jewel' the lowest. It was also reported that B- amylase is abundant and well distributed within sweetpotato root tissue. On the other hand, S-amylase activity was significantly higher in the inner than outer tissues of the roots, with the exception of 'White Delight', where no difference was observed between inner and outer tissues. S- amylase activity in the inner tissue was found to be 1.5-2.5 times higher than in the outer tissues. 'Porto Rico' had the highest S-amylase activity, and 'Jewel' the lowest. Influence of Storage on Sweetpotato Amylase Activity

Freshly harvested 'Goldrush' sweetpotatoes contained a relatively small amount of a-amylase (Ikemiya and Deobald, 1966), which increased about two-fold in three months and sixfold after nine months' storage.

Walter et al. (1975) investigated amylase activity in 'Centennial', 'Jewel', 'Porto Rico', 'Nuggett', 'Australian', 'Canner' and 'Pelican Processor' varieties. 'Pelican Processor' had the lowest a-amylase activity, while 'Centennial' and 'Porto Rico' had the highest. a-Amylase activity in 'Nugget' and 'Jewel' was always less than in

36other varieties except 'Pelican Processor'. In all cultivars, a-amylase activities increased with storage time; about 40- fold increase occurred in 'Jewel' and 'Centennial' in 71 days. S-amylase activities of the cultivars changed erratically as the season progressed. 'Centennial', 'Porto Rico' and 'Pelican Processor' were highest in IS-amylase. 'Jewel' was intermediate and 'Nugget' and 'Australian Canner' had the lowest activity.

Recently, Morrison et al. (1993) reported that a-amylase activities in 'Jewel' and 'Sumor' variety rose after harvest and reached a maximum after about 90 days. S-amylase followed nearly the same pattern, peaking at about 90 days, after which activity decreased until the last sampling date (180 days).Thermal Stability of Amylases in Sweetpotatoes

It was reported that the optimum temperature was 70-75°C for isolated sweetpotato a-amylase (Ikemiya and Deobald, 1966) and 50°C for purified S-amylase (Hagenimana et al. 1992). However, there is little published information on how long these enzymes activities can be maintained in naturally occurring conditions (in vivo). The information of stability of enzymes in vivo is very useful for controlled food processing. During enzyme purification, because of the changes of enzyme concentration and components in its solution, the stability of enzyme could be very different from that in vivo.

37DETERMINATION 07 a- AND ft-AMYLASE

A large number of valuable methods have been described for the assay of amylase. They are based on one or another of the following phenomena during enzyme digestion: (1) increase in reducing power of a solution of amylopectin or soluble starch; {2) change of the iodine-staining properties of the substrate; (3) color release from chromogenic substrates, prepared by reacting starch with organic dye. All three phenomena are characteristic for the action of a-amylases; only the first one, however, can be used for the assay of S- amylase.

It has been recognized for some time that starch and glycogen are not ideal substrates and that a more defined substrate would be preferable (Kaufman and Tietz, 1980). A method that gives a direct measure of the number of bonds cleaved in the substrate is desirable, as this can readily be converted to International Units of enzyme activity. However, assays based on the measurement of release of reducing-sugar equivalents from starch (Somogyi, 1952) are tedious and can vary, because of the nature of the starch employed and the conditions of storage of this substrate. Another problem with such an assay is that the background levels of reducing sugar in many samples results in high assay blank value.

Rinderknecht et al. (1967) established that hydrolysis of dye-bound starch by a-amylase causes a release of blue pigment (Remazolbrilliant Blue) into solution that is

38proportional over a given time interval to the amount of enzyme in the sample. This chromogenic method has found widespread use in the assay of a-amylase. It was also adapted by Walter and Purcell (1973) for investigating a-amylase levels in sweetpotato cultivars. Previous plant studies using this assay have generally assumed that this substrate is specific for a-amylase and that G-amylase is not reactive. Bilderbach (1973) demonstrated that G-limit amylopectin azure, generated by the digestion of amylopectin azure with G-amylase, would release no color by further treatment with G-amylase, but would release color upon treatment with a- amylase. He also found that the presence of G-amylase had an interfering effect on the release of color from this substrate by a-amylase. Therefore, two starch azure procedures were developed to eliminate G-amylase interference (Doehlert and Duke, 1983) : (a) the dilution procedure inwhich a serial dilution of samples to obtain G-amylase levels below levels that interfere; (b) the G-amylase saturation procedure, i.e. addition of exogenous G-amylase to increase endogenous G-amylase activity to a saturating level.

As to G-amylase, perhaps the most common method is that reported by Bernfeld (1955) . This method measures the production of reducing sugars by reaction with alkaline 3,5- dinitrosalicylic acid to form a colored complex. While this responds primarily to G-amylase, it is also affected by a- amylase activity.

39The specific determination of a- and S-amylase

activities in crude plant extracts is difficult because of the presence of both amylases in these tissues as well as the presence of free reducing sugars. A more specific, more rapid, and simpler procedure is needed.

Two methods have been developed for specific determination of a-amylase (McCleary and Sheehan, 1987) and S-amylase (McCleary and Codd, 1989). Both are based on a mixture of maltosaccharide substrate and an ancillary enzyme normally intended for a-amylase assay in human serum and urine. These substrates are chemically bonded to p- nitrophenol (PNP) through the reducing glucosyl group. Hydrolysis by the amylase, in conjunction with an a- glucosidase acting on short maltosacchrides, leads to PNP liberation, and p-nitrophenoxide formation is read at 410 nm.

The a-amylase assay substrate is p-nitrophenyl maltoheptaoside blocked at its nonreducing end (BPNPG7). S- amylase can act only after the cleavage of BPNPG7 by a- amylase, in conjunction with a-glucosidase present in the substrate. This avoids any interference by S-amylase (McCleary and Sheehan, 1987) and renders the test fully specific for a-amylase. In addition, the substrate is more stable, being resistant to hydrolysis by a-glucosidase, which is also an exo-enzyme like S-amylase. Therefore, it is possible to include this enzyme in the reagent mixture to simplify the assay procedure, i.e. two separate reactions

40take place at the same time: as soon as an oligosaccharide molecule is cleaved by ot-amylase, the action of a-glucosidase gives essentially instantaneous removal of remaining glucosyl residues releasing free p-nitrophenol. The principal of the assay is shown in Figure 2.

The S-amylase assay substrate consists of a mixture of p-nitrophenyl maltopentaoside (PNPG5) and p-nitrophenyl maltohexoside {PNPG6 ) {Mathewson and Seabourn, 1983), or uses PNPG5 only (McCleary and Codd, 1989). The principal of the assay is similar to a-amylase, as shown in Figure 3. The assay reagent comprises PNPG5 and an a-glucosidase, which has little action on PNPG5 but rapidly cleaves PNPG3. On incubation of this reagent with S-amylase, as maltose is removed by the fi-amylases, the a-glucosidase gives essentially instantaneous removal of the remaining glucosyl residues from PNPG3, releasing free p-nitrophenol. The reaction is terminated and color developed by adding Trizma base to adjust the pH to > 10. Compared to S-amylase, a- amylase cleaves these substrates very slowly, and it was shown on germinating barley, by selective inhibition studies, that a-amylase action on the test can be neglected.

These assays were compared to previous standard methods (Mathewson and Seabourn, 1983; McCleary and Sheehan, 1987). The new methods were more specific and easily adaptable to microtitration.

41

b ° T I

A o i w l o L o A - A J ' _ {L o - ’ --- ■' -O-l '--- ■■ L O J '--- ' L O J \___ / t_ 0-J L O .

Blocked p-nitrophenyl maltoheptaoside (BPNPG7)

1 Lo-/ / XN 02

O - IAlpha>amyla»

u . ' L o j ' i _ N 02o J LfOH + HCKn 'm \

Blocked maltosacchrlde p-nltrophenyl maltosaccharide

Alpha-glucocidase

/"-'“'Ax + H O -(( ) > - N 0 2

HO x 7 L OH 'v rL /

glucose p-nltrophenol

Trtim a bate

Reaction stopped and yellow color developed

Figure 2. Schematic representation of the reaction involved in the measurement of a-amylase using blocked (non-reducing glucosyl group) p-nitrophenyl maltoheptaoside in the presence of excess quantities of a-glucosidase (McCleary and Sheehan, 1987).

p-nitrophenyl maltopentaoside (PNPG5)

Beta-amylase

H(y J - J i OH

maltose

HO .'I Lo-

p-nitrophenyl maltotrloslde (PNPG3)

alpha-glucosldase

— of - AHO J x L OH

glucose

+ HO - ( l /— N02

p-nltrophenol

| Trizma baseI▼Reaction stopped and yellow color developed

N02

Figure 3. Schematic representation of the reaction involved in the measurement of 11-amylase using p-nitrophenyl maltopentaoside (PNPG5) in the presence of excess quantities of a-glucosidase {McCleary and Codd, 1989).

CHAPTER III MATERIALS AND METHODS

RAW MATERIALSThe four sweetpotato cultivars, 'Jewel', 'Centennial',

'Beauregard', and 'Hernandez' used in this study were grown at the Burden Farm of the Louisiana Agricultural Experiment Station, Baton Rouge, Louisiana following common industry practices (Broudreaux, 1991) . Roots were harvested on Oct. 20 and Oct. 26, 1994 prior to any adverse cool or wet weather. After harvest, they were cured for 10 days at approximately 30°C and 90% relative humidity, and then stored at 15°C, 85% relative humility for four months.SAMPLE PREPARATIONS AND ASSAY METHODS Enzyme AssaysPreparations of enzyme assay reagents1. Sweetpotato starch azure for a-amylase assay

A. Preparation of sweetpotato starchAbout 5000 g of Jewel sweetpotatoes were washed, hand

peeled, then chopped into approximately 0 .4-cm size in a Hobart chopper {Model 84181D, The Hobart Mfg. Co., Troy, Ohio). Chopped sweetpotatoes were blended with 25 L of water in a comminuting machine (Model D, The W.J. Fitzpatrick Company, Chicago, Illinois). The puree was filtered with two layers of cheesecloth. Filtrates were screened with a 100- mesh screen to retain cell walls not trapped by the

43

44cheesecloth (Purcell et al. 1978) . Starch grains in thefiltrates were sedimented overnight. The next day, the water was discarded, the starch was resuspended in water, screened with a 200-mesh screen, and allowed to settle by gravity. The wash water was discarded, the starch was washed with methanol (MX0485-5, EM Science, Gibbstown, New Jersey) and then with 95% ethanol (#0000517, McCormick Distilled Co., Inc., Weston, Missouri) , and finally dried in a desiccator. For a small amount of starch (e.g. 2 0 g) , it was much easier to use a home food processor and a Waring blendor instead of the Hobart and comminuting machine. Centrifugation at 1000 g for 1 0 min can also be used instead of settling by gravity.

B. Preparation of sweetpotato starch substrate labeled with remazolbrilliant blue (RBB)

Sweetpotato starch (50 g) was suspended in 500 ml of water and stirred vigorously at 50°C. A solution of 5.0 g of RBB (Sigma R-8001, Sigma Chemical Company, St Louis, Missouri) in 500 ml of water was added to the suspension. During the following 45 min, 100 g of sodium sulfate (Fisher Scientific Company, Fair Lawn, New Jersey) was added in several portions. The reaction mixture was then treated with a solution of 5.0 g of trisodium phosphate (SX0725-1, EM Science) in 50 ml of water and stirring at 50°C was continued for a further 75 min. The mixture was centrifuged and the supernatant discarded. The dark blue RBB-starch was resuspended in water and again centrifuged. Washing in this

45manner was continued until the supernatant was completely colorless. The product was now rinsed twice with methanol and dried in a vacuum desiccator over phosphorus pentoxide (Sigma P-0679) (Rinderknecht et al. 1967).2. Amylose azure for a-amylase assay

The same method as with starch azure preparation was used for amylose azure preparation except that 5 0 g of sweetpotato starch was replaced by 50 g of amylose from corn (Sigma A-7043).3. BPNPG7 for a-amylase assay

A. Preparation of phosphate buffer (0.02 M, pH 6.0) Both 1.747 g Na,HPO, (Sigma S-0875) and 10.524 g of

NaHjPO, (Sigma S-0751) were weighed and put into a 500 ml beaker. Then 300 ml of distilled water was added and solubilized facilitated with a hot plate stirrer (Model PC- 351, Corning Glass Works, Corning, New York). This solution was transferred to a 1 0 0 0 ml volumetric flask and brought to final volume of 1 0 0 0 ml by the addition of distilled water, stoppered, and mixed several times by inversion. This buffer (0.2 M, pH 6.0) was diluted 10 times to make 0.02 M phosphate buffer solution with pH 6.0.

B . BPNPG7 reagent preparationThe BPNPG7 a-amylase reagent (Sigma #577-3) is a

commercially available product from Sigma normally used to assay for a-amylase in human serum and urine. This reagent contains 4,6 -ethylidene-p-nitrophenyl-a-D-maltoheptaoside

46(ET-G7PNP or BPNPG7) which serves as the substrate for a- amylase. To prepare the reagent, 3 ml of 0.02 M phosphate buffer (pH 6.0) was added to each vial, the vial was stoppered and immediately mixed several times by gentle inversion without shaking. The final solution contained 1.0 mM BPNPG7, 10 mM MgCl,, 50 mM NaCl, a-glucosidase (25,000U/L) , and 0.05% sodium azide. Dry reagent is stable up to one year if stored at 2 -8 “C. Reconstituted reagent is stable for 14 days at room temperature (10-26°C) and 8 weeks refrigerated (2-8°C).4. PNPG5 for 6 -amylase assay

A 100 mg quantity of PNPG5 (Fluka #73734, Fluka Chemical Corp., Ronkonkoma, New York) was dissolved into 9.5 ml distilled water, mixed well, then pipetted in 0.9 ml portions to each 5 ml test tube, capped and kept in the freezer (- 20°C). Prior to the assay, one test tube was taken out of the freezer and placed into water (room temperature) to thaw. Then, 0.9 ml of distilled water and 0.2 ml with 200 unit of a-glucosidase (Sigma G-8889) were added into each test tube. Test tubes were stoppered and immediately mixed several times by inversion. Final solution contained 5 mM PNPG5 and a- glucosidase (100 U/ml) in distilled water. This reconstituted reagent was used for assaying up to 19 samples. Remaining reagent was returned immediately to the freezer for future use.

475. Soluble starch for a - and S-amylase assay

A. One percent soluble starch solutionA 1.00 g quantity of soluble starch (Sigma Prod. No. S-

2630) was weighed and placed into a 250 ml glass beaker, and then 100 ml of phosphate buffer (0.02 M, pH 6.0) was added. With constant stirring, this solution was brought to a boil on a hot plate stirrer and maintain at this temperature for 15 minutes, then cooled to room temperature. The starch solution was returned to its original volume by addition of distilled water and samples for assay were dispensed while stirring.

B. Color reagent solution for reducing sugar (Yunger,1994)

(1) Sodium potassium tartrate solutionA 12.0 g quantity of sodium potassium tartrate,

tetrahydrate, Sigma Prod. No. S-2377, was mixed with 8.0 ml of 1 M NaOH, then heated in a boiling water bath to dissolve, without allowing the solution to boil.

(2) 3,5-Dinitrosalicylic acid solutionA 438 mg quantity of 3,5-Dinitrosalicylic acid (Fluka

#42260) was weighed into 20 ml of distilled water, then heated in a boiling water bath to dissolve, without allowing the solution to boil.

(3) Color reagent solutionWith stirring, the sodium potassium tartrate solution

was slowly added to the 3,5-dinitrosalicylic acid solution.

48The mixture was diluted to 40 ml with distilled water. The solution was stored in an amber bottle at room temperature. The color reagent solution was stable for at least six months.Extraction of enzyme1. Preparation of extraction buffer

A 0.2 g quantity of CaCl,, 3 g of NaCl, and 2 g of NaN, (Sigma S-8032) were weighed into a 500 ml beaker. Phosphate buffer, 300 ml (0.02 M, pH 6.0) was added and then mixed by placing the beaker on a stir plate, using constant stirring. After the reagents were dissolved, the solution was transferred to a 1 0 0 0 ml volumetric flask, 0.08 ml of mercaptoethanol (#04 82, Amaresco, Solon, Ohio) was added and then brought to a final volume of 1 0 0 0 ml by the addition of0.02 M phosphate buffer. This extraction buffer contained 50 mM NaCl, 2 mM CaClj, 3 mM NaN,, and 1 mM S-mercaptoethanol,2. Standard extraction procedure

Randomly selected samples of six roots were used for each analysis. The unpeeled raw roots were cut in half longitudinally and one half of each root was hand-peeled, sliced into approximately 0,5-cm thick slices with a knife, then mixed by placing them in a container and shaking them in order to eliminate root variation. About 300 g of sliced samples were weighed and minced to about 1 - 2 mm size using a home food processor for 1 min. A 50 g amount of minced sweetpotato was homogenized in a Waring blendor (Model 700B,

49Waring Products Corp., Winsted, CT) for 1 min with 150 ml of ice cold (0°C) extraction buffer, and then filtered through four layers of cheesecloth. The extract was centrifuged at 13,200 G for 10 min (4°C) in a super speed centrifuge (Model RC-5B, Du Pont Instruments, Newtown, CT) , then the supernatant was collected and kept at this temperature until the enzyme assay. All assays were conducted within two hours after enzyme juice extraction.3. Crude juice (without dilution) preparation

Crude juice preparation was similar to the standard extraction procedure, with the difference that the juice was obtained by squeezing the minced roots in four layers of cheese cloth by hand, instead of using the Waring blendor and extraction buffer.Assay of q-amvlase1. Starch azure method

Enzyme assays of the juice were run in duplicate. Substrates were prepared from sweetpotato starch Azure at 2% level using extraction buffer, following procedure as described by Walter and Purcell (1973). The substrate was swirled until a homogenous suspension was obtained and then a 2.7 ml aliquot was rapidly pipetted into 15 ml centrifuge tubes. These tubes were placed in a water bath (Cat No. 66799, Precision Scientific Group, Chicago, Illinois) at the reaction temperature 60°C for 15 min. Crude juice samples (0.3 ml) warmed to room temperature were pipetted into each

50tube. Tubes were stoppered and mixed several times by inversion, and a timer started. After exactly 15 min, the reaction was stopped by adding 1.2 ml of 5% v/v trichloracetic acid {#2928, Mallinckrodt, Inc., Paris, Kentucky) followed by vigorous mixing. Blanks were prepared by adding trichloracetic acid solution to the substrate prior to the juice sample addition. After stopping the reaction, the tubes were centrifuged for 3 min in a clinical centrifuge at speed 5 {Model CL, International Equipment Co., Needham Hts., Mass.) and filtered through Watman #2 paper. Absorbance was then measured at 595 nm in a spectrophotometer (Series Du-65, Beckman Instruments, Inc., Fullerton, CA). The calculations are as follows:Sweetpotato a-amylase activity on starch azure

_ iAs9s „ dilutionUnit / ml Asss 0f o.iftf CuSOt factor

AA59S = A (reaction) - A (blank)

The 0.1 M copper sulfate (#4844, Malinckrodt) solution was used as an arbitrary standard so that results using different spectrophotometers could be compared.2. Amylose azure method

The procedure described for starch azure assay was used except that amylose azure substrate was used instead of sweetpotato starch azure.

513. BPNPG7 standard assay method

A 0.2 ml of BPNPG7 a-amylase reagent was pipetted into a 5 ml test tube and warmed for 2 min in a 4 0°C water bath. Then, 0.1 ml of enzyme preparation from standard extraction procedure (warmed to room temperature} was immediately added and incubated with the reagent at 4 0°C for exactly 10 min without shaking. The reaction was terminated and color developed by adding 3 ml of 1% (w/v) Trizma base (Sigma T-1503) and the absorbance of the solution was then determined at 410 nm in a spectrophotometer. The blank was prepared following the same procedure except that 0 . 2 ml of distilled water was used instead of the amylase reagent. One unit of enzyme was defined as the amount of enzyme that releases 1

fimole of p-nitrophenol/min under defined assay conditions (McCleary and Sheehan, 1987}Calculation of activity:

Sweetpotato a-amylase activity (U/g fresh sample) =A A 4io ^ total assay vol. 1 „ extraction „ j,-,,,.. „ --- .---,---- ,-- x — --------£_---r x ___ x x dilution.incubation time aliquot assayed volume

Where AA,10 = A (reaction) - A (blank) , in this study,incubation time = 10 min, total assay volume in cell = 3.3 ml, aliquot assayed - 0 . 1 ml, E*, (molar extinctioncoefficient of p-notrophenol) = 16.6 ml/itmol, extractionvolume *= 4 ml/g fresh sample, dilution = 1.The calculated activity would be given by

52

{Unit/g)1 0 min 0 .1 ml^ 4 1 0 3.3 ml

■ * . ■ 16.6 ml/fxmol 9= A A x 0.795

Assay of S-amvlase1. Bernfeld (1955) method

Activity of S-amylase was determined by using 1% soluble starch in phosphate buffer (pH 6.0) as the substrate. A 0.1 ml sample of juice or dilution was added to 0.9 ml of substrate. The reaction was continued for 10 min at 40°C, then stopped with 0.5 ml color reagent. The mixture was heated in boiling water bath for 5 min and cooled on ice. Three ml of distilled water was added for dilution. The amount of reducing sugar produced was determined as maltose at 54 0 nm, and one unit of enzyme was expressed as mg of maltose per min under defined conditions.2. PNPG5 standard assay method

The enzyme juice obtained from standard extraction procedure was further diluted 4000 times with 20 mM phosphate buffer (pH 6.0) containing 0.3% NaCl, 0.02% NaN,, 0.037% Di- NaEDTA (Fluka, 03679), 1 mg/ml BSA (Sigma A-4503), pH 6.0. A 0.1 ml aliquot of PNPG5 S-amylase reagent was pipetted into a 5 ml test tube and warmed for 2 min in a 4 0°C water bath. Then 0.1 ml of the dilution was added and incubated with this reagent at 40°C for exactly 10 min. The reaction was terminated and color developed by adding 3 ml of 1% (w/v)Trizma base and the absorbance of the solution was then

53determined at 410 nm in a spectrophotometer. One unit of enzyme was defined as the amount of enzyme that releases 1 fimole of p-nitrophenol/min under defined assay conditions (McCleary and Codd, 1989).Calculation of activity

Sweetpotato 0-amylase activity {U/g fresh sample) =

_ AA“ “ * total assay yol x _ 1 _ x extraction x dilutionincubation time aliquot assayed E^ volume

Where AA ,, = A (reaction) - A (blank) , in this study,incubation time = 10 min, total assay volume in cell = 3.2 ml, aliquot assayed = 0.1 ml, E*, (molar extinctioncoefficient of p-notrophenol) = 16.6 ml//imol, extractionvolume = 4 ml/g fresh sample, dilution = 4000.The calculated activity would be given by

(Uni t/g) = ^A41,° x 3 ‘ * x x x 400010 m m 0.1 ml 16.6 ml/pmol g

= A A x 3004.3

P r o t e i n D e t e r m i n a t i o n

Total proteins were quantified by the method of Bradford (1976), with BSA as standard.Preparation of protein reagent

One hundred milligram of Coomassie Brilliant Blue G-250 (Sigma B-0770) was dissolved in 50 ml 95% ethanol. To this solution 100 ml 85% (w/v) phosphoric acid (Mallinckrodt#2796-8) was added. The resulting solution was diluted to a final volume of 1000 ml. Final concentrations in the reagent

54were 0.01% (w/v) Coomassie Brilliant Blue G-250, 4.7% (w/v) ethanol, and 8.5% (w/v) phosphoric acid.

The sweetpotato juice obtained from standard extraction procedure was diluted 10 times by distilled water. A 0.1 ml of this dilution was pipetted into a 5 ml test tube and 3 ml of protein reagent was added. The absorbance at 595 nm was measured after 2 min and before 1 hr in 3 ml cuvettes against a reagent blank prepared from 0.1 ml of 0.002 M extraction buffer and 3 ml of protein reagent. The protein contents in unknown samples were calculated from the linear equation of standard curves.Protein standard

A BSA protein standard was obtained from Sigma {P 5304} and was diluted to different concentrations (80, 60, 40, 20, 10, 5 ^g protein/ml) by 0.002 M extraction buffer. The same procedure was followed as used in the sample assay; the absorbances corresponding to the different concentrations of protein were measured. Based on these data, a linear equation was obtained, which could be used for calculation of protein contents in samples. In this study, the linear equation was: Y = 94.92 X - 4.23. Where Y is protein concentration insample (jig/ml) , X is absorbance at 595 nm.Dry Matter Determination

The same chopped sweetpotato sample prepared for enzyme extraction was used for dry matter determination. Duplicate 10-g samples were weighed to 1 mg, dried for 4 8 hr at 70 °C in

55a forced-air oven {Model no. OV-490A-2, Blue M, Blue Island, Illinois) removed, allowed to cool for 3 0 min in a desiccator, and again weighed (Picha, 1985) .Puree Preparation

The same chopped sweetpotato sample prepared for enzyme extraction was used for puree preparation. One hundred gram of the chopped material was mixed with 100 ml distilled water in a 500 ml beaker with tare weight recorded, and rapidly heated to 80°C with stirring by a thermometer in about 5 minutes in a boiling water bath, samples were then covered with foil and kept at this temperature for 30 min in an 80°C water bath (Model MW-1120C-1, Blue M Electric Company, Blur Island, Illinois). After heat treatment, the reaction was stopped by immediately microwave heating (high power level-1 min, low power level-5 min.), and samples were cooled to room temperature. The amount of water loss during heating was calculated by subtracting the tare weight from the total weight. Then distilled water was added to bring the sample to the original weight (200 g). The sample was homogenized in a Waring blendor for 1 min. Puree from each of the products was analyzed for total solids, alcohol insoluble solids, sugar content and apparent viscosity.Determination of Viscosity

Apparent viscosity was measured using a Brookfield Digital Viscometer (Model DV-II, Brookfield Engineering labs,

56Inc., Stoughton, MA) with a spindle number A of the HB type at 10 rpm for 3 min, at 25°C.Determination of Alcohol Insoluble Solids (AIS)

Ten g of the same chopped fresh roots prepared for enzyme extraction or puree was homogenized in 80% ethanol for 1 min at high speed using a Virtis 45 homogenizer {The Virtis Co., Inc. Gardiner, New York) . The resulting slurry was immediately boiled for 15 min, cooled, and filtered through Whatman #4 paper. The residue and original container were washed with an additional 00V ethanol and the filtrate was made to a final volume of 100 ml. AIS content was determined by the weight of the insoluble residue retained on the filter paper after 24 hr of drying at 50°C under vacuum (Picha, 1987) .Sugar Datermination

The filtrate obtained from the AIS procedure was made to a final volume of 100 ml with 80V ethanol. Sugars were determined by HPLC (Picha, 1987) and the method described by Bernfeld (1955) .Sugar analysis bv HPLC

An aliquot was taken by a 5 ml syringe (#1603, Becton Dickinson and Company, Frankling Lakes, New Jersey) and filtered into 1 ml scintillation vials (223682-CMS, Wheaton Scientific, Millville, New Jersey) through a Nalgene syringe filter with 0.45 pm membrane (#199-2045, 25 mm, Nalge

57Company, Rochester, New York), then capped with teflon lined crimpcaps for injecting into the HPLC.

A Beckman liquid chromatograph (series 340, Beckman Corporation, Fullerton, CA) equipped with a pump (model 112) , an injector (model 210) fitted with a 20/*L sample loop and a refractive index detector (model 156) was used. The detector signal was electronically integrated by a Varian 401 integrator in the external standard mode using an attenuation of 16 and a chart speed of 0.5 cm/min. Sugars were separated with a 3 00 mm x 7.8 mm i.d. column packed with Aminex HPX-87C resin (Bio-Rad Labs, Richmond, CA) heated to 75°C. Plumbed between the injector and the analytical column were: a 2m Rheodyne 7302 column inlet filter and a 40 x 4.6 mm ion exclusion guard cartridge packed with Aminex A-25 resin (both from Bio-Rad Labs). The mobile phase was degassed HPLC grade acetonitrile (NA 1648, Mallinckrodt, Inc.) solution containing 30V deionized distilled water having a flow rate of 1.2 ml/min. During operation of the HPLC, for every 8 samples of sugar, one sample of standard sugar solution was injected in order to check the accuracy of the HPLC.

The sugar standard containing IV glucose, 1% fructose, 4V sucrose, and 8% maltose, was prepared by adding 1.000 g of glucose (Sigma G-7528) , 1.000 g of fructose (Sigma F-0127),4.000 g of sucrose (Amresco, Solon, Ohio), and 8.000 g of maltose (Sigma M-5885) to 80% ethanol and boiling, following the same procedure as for sample preparation. The standard

58solution was filtered, made up to 100 ml with 80V ethanol and filtered before injecting into the HPLC.Determination of reducing sugar

Reducing sugar was determined by the method of Bernfeld (1955) . After the filtrate was made up to 100 ml with 80V ethanol, a 0.4 ml aliquot was pipetted into each test tube with 1.6 ml distilled water. One ml of reducing sugar color reagent was added and the mixture was placed in a boiling water bath for exactly 5 min, cooled on ice and nine ml distilled water was added. Absorbance was measured at 54 0 nm for both the sample and blank.Starch AssayPreparation of amvloglucosidase digestion solution

Two g of amyloglucosidase (Sigma A-7255) was mixed into l L of ice-cold 0.05 M sodium citrate buffer, pH 4.5. This solution provided enzyme activity in excess of that required to attain complete starch digestion for any plant tissue. As supplied by Sigma, this chemical contained approximately 40V diatomaceous earth, 33V protein, 5V water, and 22V starch/sugar. The starch and diatomaceous earth were removed by centrifuging this chemical at 13,200G for 5 min immediately after mixing. This procedure eliminates high blank value (Rose et al. 1991).Starch assay procedure

About 15 mg of AIS samples was weighed into 5 ml test tubes, 2 ml of distilled water was added, and the samples

59were heated for 1 hr in a covered boiling water bath. After cooling, 2 ml of amyloglucosidase digestion solution was added to each sample. The samples were incubated at G0°C for 1 hr. Then 0.1 ml of each solution was added to 0.9 ml of distilled water in 5 ml test tubes, A 0.5 ml aliquot of reducing sugar reagent was pippeted into the test tube, the mixture was heated in a boiling water bath for 5 min, and cooled on ice. A 1.5 ml aliquot of distilled water was added for dilution. The amount of reducing sugar produced was determined as glucose by measuring absorbance at 54 0 nm. A blank was prepared following the same procedure as sample preparation except that 2 ml of amyloglucosidase digestion solution was inactivated by boiling for 10 min, which was then added to the sample for further incubation. Final starch content in AIS was calculated by the total amount of glucose produced during conversion multiplied by the starch hydrolysis factor 0.9 (Rose et al. 1991).STATISTICAL ANALYSIS

The data were subjected to ANOVA (analysis of variance) and regression determination using the SAS Statistical Package (SAS institute, Cary, N.C.). Differences were considered significant when means of compared sets differed at the p<0.05 level of significance.

60EXPERIMENTAL DBSION AND PROCEDUREExperiment 1. Assay of a - end ft-Amyleae Activity

All treatments in this experiment were conducted in triplicates. All enzyme concentrations used in this experiment were determined in preliminary experiments and the concentrations were within suitable assay ranges. Standard extraction buffer was used for a-amylase dilution. Phosphate buffer {0,02 M, pH 6.0) containing 0.3% NaCl, 0.02% NaNj,0.037% Di-NaEDTA, BSA 1 mg/ml was used for S-amylase dilution.Analytical approach

The objective of these experiments was to adapt two newly developed methods for amylase assays in sweetpotatoes to eliminate interference between a- and S-amylase. It was necessary to meet certain criteria in the development of assays with crude extracts; therefore, linearity, specificity, sensitivity, and correlation with traditional methods were evaluated.Linearity of BPNPG7 a-amvlase assay procedure

Alpha-amylase from Bacillus subtilis (#100447, ICN Biomedical Inc., Aurora, Ohio) was diluted 50,000 times, then further made into a series of dilutions that contained a- amylase at 2, 1.6, 1.2, 0.8, 0.4, 0.2 mg/100 ml. A 0.1 ml of each dilution was pipetted into 0.2 ml BPNPG7 a-amylase reagent and assayed following standard procedure. Correlation coefficient between concentration and corresponding

61absorbance was determined by linear regression program of SAS.

Linearity of native a-amylase levels with absorbances was also determined following the same procedure as used with commercial a-amylase. The crude sweetpotato juice obtained from 'Beauregard' was diluted to different concentrations (20%, 16%, 12%, 8%, 4% and 2%) . A 0.1 ml aliquot of eachdilution was pipetted into 0.2 ml BPNPG7 a-amylase reagent and assayed following standard procedure. Linearity of the reaction with time was also determined by incubating 0.1 ml of 8% crude juice with 0.2 ml BPNPG7 a-amylase reagent in six test tubes. Reaction was stopped after 5, 10, 15, 20, 25, 30 min of incubation. The absorbances were measured and their correlation with incubation times was determined.Linearity of PNPG5 fi-amvlase assay procedure

Beta-amylase from sweetpotato (Sigma A-7005) was diluted50,000 times, then further made into a series of dilutions that contained fi-amylase at 20, 16, 12, 8, 4, 2 /il/L. A 0.1 ml aliquot of each dilution was pipetted into 0.l ml PNPG5 £- amylase reagent and assayed following standard procedure. Correlation coefficient between concentration and corresponding absorbance was determined by linear regression program of SAS.

Linearity of native S-amylase levels with absorbances was also determined following the same procedure as for commercial S-amylase. The 'Jewel' sweetpotato juice obtained

62from standard extraction procedure was diluted 1000 times, then further made into a series of dilutions that contained juice extract made up to 100, 75, 50, 25, 10, 7.5, 5 fil/L. A0.1 ml aliquot of each dilution was pipetted into 0.1 ml PNPG5 6-amylase reagent and assayed following standard procedure. Linearity of the reaction with time was also determined by incubating 0.1 ml of 25 fil/L juice dilution with 0.1 ml PNPG5 fi-amylase reagent in six test tubes. Reaction was stopped after 5, 10, 15, 20, 25, 30 min ofincubat ion. The absorbances were measured and their correlation with incubation times was determined. Reproducibility

Reproducibility was analyzed to check the reliability of the assays. The a- and fi-amylase activities in four sweetpotato cultivars were assayed eight times for each cultivar within the same day by standard assay procedures (starting with extraction) to determine the precision of the a- and fi-amylase assay procedure. Standard deviation and coefficient of variation (c.v.) of final assay results within each cultivar were determined.Specificity of substrates BPNPG7 and PNPG51. Action of S-amylase on BPNPG7 a-amylase reagent

Sigma fi-amylase was diluted to 10, 100, 1,000, 10,000,and 100,000 times, then 0.1 ml of each dilution was added to0.2 ml BPNPG7 a-amylase reagent and assayed following standard procedure. To investigate the effect of fi-amylase on

63BPNPG7 in the presence of a-amylase, 1 ml of 1/50,000 of ICN a-amylase was mixed with 1 ml of each dilution of S-amylase (from 10 to 100,000 times). Then, 0.1 ml of each mixture was incubated with 0.2 ml BPNPG7 a-amylase reagent and assayed following standard procedure.2. Action of a-amylase on PNPG5 fi-amylase reagent

Alpha-amylase from ICN was diluted to 50,000 times, thenfurther made into a series of dilutions that contained a-amylase at 2, 1.6, 1.2, 0.8, 0.4, 0.2 mg/100 ml. A 0.1 mlaliquot of each dilution was pipetted into 0.1 ml PNPG5substrate mixture and assayed following standard procedure.At the same time, the activities of the dilutions on BPNPG7were also assayed following standard procedure.Comparison of traditional and new methods for a-amylase and fi-amylase assay

A series of dilutions (2, 1.6, 1.2, 0.8, 0.4, 0.2 mg/100 ml) of a-amylase from ICN were assayed by standard BPNPG7 procedure. Another series of dilutions (20, 16, 12, 8, 4, 2mg/100 ml) of a-amylase from ICN were assayed by starch azure methods. Correlation coefficients obtained by comparison of these two methods were determined by linear regression of SAS. Sensitivities were also compared based on a certain amount of enzyme activity change corresponding with absorbance unit change under defined conditions. Following the same procedure, the BPNPG7 assay method was compared with the Amylose method and the Bernfeld method, respectively.

64For E-amylase assay methods comparison, a series of

dilutions (20, 16, 12, 8, 4, 2 fil/h) of E-amylase from Sigma were assayed by standard PNPG5 procedure. Another series of dilutions (100, 80, 60, 40, 20, 10 (il/L) of E-amylase fromSigma were assayed using the Bernfeld method. Correlation coefficient between these two methods was determined by linear regression of SAS. Sensitivities were also compared based on a certain amount of enzyme activity change corresponding with absorbance unit change under defined conditions.Determination of Km and Vmax

Michaelis-Menten constant (Km) and substrate turnover number (Vmax) were determined by linear regression of the Lineweaver-Burk plot (Lineweaver and Burk, 1934). Kinetic data with BPNPG7 as a substrate for Of-amylase reaction were obtained with substrate concentrations of 2.0, 1.0, 0.8, 0.5, and 0.4 mM at pH 6.0 at a temperature of 4 0°C. Substrate concentration of PNPG5 for fi-amylase reaction were 5.0, 2.5, 2.0, 1.25, 1 mM, with reaction conditions of pH 6.0 and 40°C. Temperature effects on a- and fi-amvlase assay

The sweetpotato juice from 4 cultivars was prepared as standard enzyme extraction procedure, a- and fi-Amylase activity of the juice was assayed at 30, 40, 50, 60, and 70°C by standard BPNPG7 and PNPG5 procedure, except that warming time for reagents was changed from 2 min to zero min, also,

65juice from Centennial was diluted 8,000 times instead of 4 , 000 times.Experiment 2. Characterization of Some Properties of Sweetpotato AmylasesAnalytical approach

These experiments were performed to characterize the stability of a-amylase and 6-amylase in vivo, which is a valuable factor to be considered in sweetpotato processing, enzyme preservation, and enzyme assay. In addition, the effect of the combined action of a-amylase and 6-amylase was also investigated.Temperature effecfc__pn stability of amylases

'Beauregard' sweetpotato juice was prepared as in the standard procedure except that distilled water was used instead of buffer for enzyme extraction in order to eliminate any effect of chemicals. A 0.5 ml aliquot of the juice was pipetted into 56 five ml test tubes, capped and placed in different temperature conditions {-20, 4, 25, 75°C) with 14 test tubes for each temperature level. For -20°C and 4°C treatment, a 0.1 ml of juice was taken from each test tube after 0, 1, 4, 6, 16, and 28 days, and the remaining a- and6-amylase activity was assayed by standard BPNPG7 and PNPG5 procedures. For 25°C treatment, 0.1 ml of juice was taken from each test tube at 0, 12, 24, 3 6 hr, 4 days and 6 days. For 75°C treatment, 0.1 ml of juice was pippeted into 0.2 ml BPNPG7 a-amylase reagent atO, 0.5, 1, 1.5, 2, 3, 4, and 5min, and the reaction was started immediately (blanks for a-

66a-amylase assay were taken at the same time interval) . A 0.1ml aliquot of juice was taken from each test tube at 0, 5,10, 15, 20, 25, and 30 min for fi-amylase assay.Dilution effect on the stability of amylases

'Beauregard' roots were hand peeled, grated, and juiceequivalent to 23-25% of the weight was obtained by squeezingthe grated roots in a Shear press. The juice was centrifugedat 13,200 g for 10 min (4°C) and the supernatant collected.Dilutions of 2, 4, and 8 times were made by adding distilledwater. A 0.5 ml aliquot of each dilution was pippeted into 7test tubes, capped and placed into a 758C water bath. Foreach dilution level, a 0.1 ml sample was taken from each testtube at different times {similar to the 75°C treatmentdescribed previously) and the remaining a- and 6-amylaseactivity was assayed by standard BPNPG7 and PNPG5 procedures.Comparison q£ Lb£ stability of amvlases in differentcultivars

The sweetpotato juice from 4 cultivars was prepared as in the standard enzyme extraction procedure except that distilled water was used instead of buffer for enzyme extraction. A 0.5 ml aliquot of the juice was pipetted into a 5 ml test tube, capped and placed into 75°C water bath. The remaining a- and fi-activity at different heating times (similar to the 75°C treatment described previously) was assayed by standard BPNPG7 and PNPG5 procedures.

67Interaction of commercial a-amylase and fi-amylase on atarch hydrolysis.1. Interaction at normal assay concentration

A 1/100,000 dilution of a-amylase from ICN (A) and 1/10,000 dilution of B-amylase from Sigma (B) were further diluted to 80, 60, 40, 20, 10% of each concentration.Mixtures of a and fi-amylase with different dilutions were prepared by mixing equal amounts of a- and fi-amylase solution with the same dilution level. For example, 0.2 ml of 80% A was mixed with 0.2 ml of 80% B; 0.2 ml of 40% A was mixed with 0.2 ml of 40% B . The total reducing power of each preparation was assayed by the Bernfeld method with 1% gelatinized sweetpotato starch in phosphate buffer (pH 6.0) as substrate.2. Interaction at high concentration

The same procedure was followed as described in the previous section except that a 1/10,000 dilution of a-amylase and 1/1,000 dilution of fi-amylase were used. The assay method was modified as follows: after 10 min reaction at 40°C, 0.1 ml of sample was pipetted into 0.5 ml color reagent to stop the reaction and 0.9 ml distilled water was added. The mixture was heated in a boiling water bath for 5 min and cooled on ice. Three ml of distilled water was added for dilution. The amount of reducing sugar produced was determined as maltose at 540 nm.

68Experiment 3. Amylases Activity and Carbohydrate Changes in Sweetpotatoes during Storage and Their Effects on Puree ViscosityAnalytical approach

The objective of these experiments was to investigate the major causes of inconsistent products of sweetpotato puree and provide useful information in establishing optimal procedure to improve inconsistency in sweetpotato processing. Since puree processing is actually an enzymatic hydrolysis procedure, there are two possible factors that could contribute to inconsistency, i.e. enzyme level changes and carbohydrate (substrate) changes during storage. These changes were evaluated.Methods

The sweetpotatoes of four cultivars harvested on Oct. 20, 1994 and Oct. 26, 1994 were used as two differentreplications in this study. Raw roots from each cultivar were used on the day of harvest, after curing, and 1, 2, 3, 4months after harvest. Six random raw roots from each cultivar were prepared for analyses. All the assays for each sample were run in duplicate. Analyses for each fresh sample included a-amylase (BPNPG7 assay procedure), S-amylase (PNPG5 assay procedure), dry matter, AIS, reducing sugar, sucrose (HPLC), and protein content. For purees prepared from each fresh sample, dry matter, AIS, maltose (HPLC), and viscosity were determined.

CHAPTER IV RESULTS AMD DISCUSSION

EXPERIMENT 1. ASSAY OF a- AND S-AMYLASE ACTIVITY Linearity of BPNPQ7 a-Amylase Assay Procadura

In principle, enzyme activity can be assayed by measuring the changes in the concentration of product. Therefore, one of the basic requirements for an enzyme assay method is that the concentration of enzyme preparation must be directly proportional to the amount of the product formed during reaction. The linearity of the reaction of BPNPG7 with enzyme concentrations was determined by incubating 0.1 ml commercial a-amylase obtained from ICN (#100447) and sweetpotato juice at different concentrations with 0.2 ml standard BPNPG7 substrate mixture. Both tests show that their activities correlate very well with their concentrations up to 0.6 absorbance unit (or equal to 120 mU/ml), with correlation coefficient r = 0.9966 and 0.9975, respectively (Figure 4 and Figure 5). Unlike commercial a- amylase, the sweetpotato juice is a crude homogenate. A control (0.1 ml juice, 0.2 ml reagent, and 3 ml Trizma base) should be used to obtain accurate results, otherwise, nonlinearity could be found in the response pattern. However, the reagent blank (0.1 ml extraction buffer, 0.2 ml reagent, and 3 ml Trizma base) is very stable and has little effect on

69

AB

SOR

BA

NC

E

(410

n

m)

0.6

0.5

0.4

0.3

0.2

0.1

OO 2 4 6 8 lO 12 14 16 18 20

CONCENTRATION (%)

Figure A. Linearity of the BPNPG7 a-amylase assay with commercial a-amylase concentration. - jo

0.6

C O .S

0.4

0.3

0.2

0.1

18 20

CONCENTRATION (%)

Figure 5. Linearity of the BPNPG7 a-araylase assay with concentration of extractedsweetpotato juice. Sweetpotato juice was squeezed from grated Beauregard roots anddiluted using distilled water.

72the absorbance (from 0.003 to 0.005). Therefore, for economy, 0.2 ml of distilled water could be used for the control instead of reagent. In order to measure the rate of reaction, a sufficient amount of product must accumulate for accurate measurement. During the course of accumulation, the rate of the reaction must remain constant. Time-reaction course studies showed that a-amylase activity in sweetpotatoes is linearly related to reaction time through 30 min with r = 0.9985 (Figure 6). Eventually, the rate of reaction became slower, due to depletion of substrate or inhibition by product. This may be overcome by increasing the substrate concentration or decreasing the amount of enzyme added. Linearity of PNPG5 &-Amylase Assay Procedure

The linearities of the reactions with commercial fi- amylase and sweetpotato extract concentrations are shown in Figure 7 and Figure 0. At assay temperature of 40°C, linear kinetics are observed up to 0.90 absorbance unite (or 173 mU/ml) , with r = 0.9975 and 0.9980. Since the G-amylase level in sweetpotatoes is extremely high, the juice extract needs to be further diluted 4000-fold to conduct the standard assay procedure, fi-amylase is quite stable in crude sweetpotato extract (results presented later). However, when highly diluted, i.e., to a level suitable for assay, the enzyme is unstable. It was found that stabilities considerably increased by adding cysteine (20 mM) , mercaptoethanol (1 mM) or dithiothreitol (l mM) to the buffer, but by far the best

AB

SOR

BA

NC

E

(410

n

m)

0.6

0.5

0.4

0.3

0.2

0.1

O 5 lO 2015 30

TIME (min)

Figure 6. Linearity of the BPNPG7 a-amylase assay with incubation time of a 1/2dilution of sweetpotato extract. The sweetpotato extract was prepared from Beauregardroots using standard extraction procedure.

AB

SOR

BA

NC

E

(410

n

m)

1.2

0.8

0.6

0.4

0.2

lOO 5 25 3015 20CONCENTRATION 0*L/L)

Figure 1. Linearity of the PNPG5 fl-amylase assay with commercial sweetpotato fi-amylase concentration.

AB

SOR

BA

NC

E

(410

n

m)

0.8

0.6

0.4

0.2

15050 1 0 0 200 250

CONCENTRATION OiUL)

Figure 8. Linearity of the PNPG5 fl-amylase assay with concentration of extracted sweetpotato juice. Sweetpotato juice was extracted and assayed using standard procedure. Ln

76stabilizer was BSA at 1 mg/ml. In the presence of this concentration of BSA, there was essentially no loss in activity on storage of the diluted enzyme at 22°C for 2 h (McCleary and Codd, 198 9). Therefore, a 2 0 mM phosphate buffer (pH 6.0) containing 1 mg/ml BSA was used for the dilution of sweetpotato S-amylase. After such a high level dilution, the juice sample has no effect on blank absorbance, thus there is no need to prepare juice blank for each sample, only reagent blank (0.1 ml regent, 0.1 ml buffer for dilution, 3 ml Trizma base) is needed. The linearity of the S-amylase activity in the sweetpotatoes for the hydrolysis of PNPG5 with time is shown in Figure 9. The enzyme activity is linearly related to reaction time up to 30 min with a correlation coefficient r = 0.9997. The reaction isstoichiometric and the reaction curve passes through the origin.Reproducibility

Reproducibility was analyzed to check the reliability of the assays. The or- and fi-amylase activities in four sweetpotato cultivars were assayed eight times for each cultivar within the same day by standard assay procedures (starting with extraction) to determine the precision of the a- and fi-amylase assay procedure.

Statistical analysis gave a 2.89% of c.v. (coefficient of variation) for the a-amylase assay and 4.07% of c.v. for fi-amylase assay (Table 3).

AB

SOR

BA

NC

E

(410

n

m)

0.8

r= 1.000

3025201510TIME (min)

Figure 9. Linearity of the PNPG5 fl-amylase assay with incubation time of a 1/8000dilution of the sweetpotato extract. The sweetpotato extract was prepared fromBeauregard roots using standard extraction procedure.

Table 3. Reproducibility of the BPNBG7 and PNPG5 assay for the measurement of sweetpotato a-amylase and S-amylase.

SAMPLE a-AMYLASE ACTIVITY (U/g fresh sample) Average C.V%

BEAUREGARDCENTENNIALHERNANDEZJEWEL

0.3590.4090.3060.208

0.3630.4050.3040.209

0.3630.3900.3030.203

0 .359 0.388 0 .299 0 .204

0.3760.3900.3150.213

0.370 0.362 0.393 0.421 0.310 0.305 0.216 0.209

0.3780.4410.3200.219

0.3660.4050.3080.210

2.044.622.242.89

Average C.V 2.89.

SAMPLE S-AMYLASE ACTIVITY (U/g fresh sample) Average C.V*

BEAUREGARDCENTENNIALHERNANDEZJEWEL

607.6 2461 727. 9 749. 5

613 .8 2405 653.9 715.6

660.0 2587 721.7 808.1

653 . 9 2556 715.6 811.2

657.0 2581731.0 869.8

647.7 641.5 2498 2415 709.4 737.1 851.3 845.1

629.2 2458 724.8 838. 9

638.82495715.2811.2

3 .12 2.90 3.67 6.58

Average C.V.%: 4.07.

For each sample, eight separate assays were made using standard procedure within three hours.

-j<D

79

Specificity of Substrates BPNPG7 and PNPG5(A) Action of 15-amylase on BPNPG7

The effect of 15-amylase on a-amylase substrate BPNPG7 is shown in Table 4. The BPNPG7 had no response to 15-amylase with activity from 0.05 to 500 PNPG5 Units. In the presence of a-amylase, S-amylase with different concentrations did not change the a-amylase assay results. It is evident that the BPNPG7 is totally specific for a-amylase, providing the ability to measure this activity specifically even in the presence of a concentrated commercial 15-amylase (50,000-fold excess of normal assay concentration).(B) Action of a-amylase on PNPG5

The action of a-amylase on PNPG5 and BPNPG7 is shown in Table 5. The PNPG5 substrate is approximately thirty times more resistant to hydrolysis by a-amylase than is BPNPG7. However, PNPG5 is not completely specific for S-amylase, it could affect final reading for S-amylase assay. The degree of interference by a-amylase varies depending on its concentration. Although PNPG5 substrate is not as specific as BPNPG7, it is still possible to calculate the level of S- amylase accurately in a given sample.

The action of a-amylase on PNPG5 and BPNPG7 produced different results. However, it was found that there was a linear relationship between their activities with correlation coefficient r - 0.994. Therefore, the degree of effect of a-

80

Table 4. Action of S-amylase on a-amylase assay substrate BPNPG7.

S-Amylase activity PNPG5 U/ml

S Amylase onlyAbsorbance1

S-Amylase plus a-amylase

Absorbancea-AmylaseonlyAbsorbance

0 . 050 0 . 004 0 .362 0 .3540.498 0 . 009 0 . 345 0 .3544 . 975 0 . 004 0 . 346 0 .35449 .75 0 .007 0 .351 0 .354497.5 0 . 004 0 .365 0 .3541 Absorbance was determined at 410 nm in aspectrophotometer. Reaction condition was 0.2 ml ofBPNPG7 reagent and 0.1 ml of various enzyme preparations (prepared as described under Materials and Methods) at4 0 °C for 10 min and pH 6.0.

81

Table 5. Action of a-amylase on fi-amylase assay substrates PNPG5 and soluble starch.

Activity BPNPG7 PNPG5 BernfeldU BPNPG7/ml Absorbance1 Absorbance3 Absorbance5

0 . 0165 0 . 003 0 . 006 0 .1520 . 0314 0 .158 0 .010 0 .4200 . 0580 0 .292 0 .015 0 .9150 . 0811 0.408 0 .018 1. 4130 .0998 0 . 502 0 . 021 1 . 7780 .1149 0 . 578 0 . 023 2 . 137Sensitivity 5 .030 0 . 168 20.099(AABS/1 u BPNPG7 change)1 Absorbance (ABS) was determined at 410 nm in aspectrophotometer using standard BPNPG7 assay method.1 Absorbance was determined at 410 nm in aspectrophotometer. Reaction condition was 0.1 ml of PNPG5 reagent and 0.1 ml of various a-amylase preparations (prepared as described under Materials and Methods) at 40°C for 10 min and pH 6.0.5 Absorbance was determined at 540 nm in a spectrophotometer using standard Bernfeld assay method.

82amylase on fi-amylase assay can be calculated by the linear equation: B = 0.003 + 0,032 A (derived from data in Table 6). Where A is a-amylase activity on BPNPG7, B is a-amylase activity on PNPG5. In the mixture of a- and fi-amylase, the exact 6-amylase activity can be determined by assaying the extract on PNPG5 to give apparent 6-amylase activity, and then subtracting from a-amylase activity on PNPG5. Therefore, fi-amylase (U/g sample) = apparent activity - a-amylase activity on PNPG5. By using the linear equation above, fi- amylase activity = apparent activity - [0.003 + 0.032 x a-amylase activity on BPNPG7].

The ratio of activity of BPNPG7 and PNPG5 was used by McCleary and Codd (198 9) instead of the linear equation. Since the ratio is not consistent, it could vary from 15 to 3 0 depending on enzyme concentration (Table 6), the linear equation can more accurately determine the degree of effect of a-amylase.

McCleary and Codd (1989) noticed the differences in the ratios of activity of a-amylases from different sources. It is possible that the linear equation could be different, as well. Thus, the linear equation should be determined for a- amylases from different sources.

Sweetpotatoes contain abundant S-amylase, the extract requires 4,000-fold dilution before assay. The a-amylase activity in sweetpotato extract ranges from as little as 0.05 to 0.10 U/ml. After such a dilution, the activity on

83BPNPG7 should be between 0.0125 and 0.025 mU/ml. The effecton PNPG5 should be much lower than that. The activity ofsweetpotato S-amylase on PNPG5 in assay aliquot ranges from3 80 to 1540 mU/ml, which is much higher than 0.01 mU/tnl.Therefore, it can be concluded that the effect of theendogenous sweetpotato a-amylase on the specificity of thePNPG5 E-amylase assay in sweetpotato is negligible.Comparison of Traditional and Haw Methods for a-Amylase and S-Amylase Assay.

The traditional amylase assay procedures are widely used in cereal, food and fermentation industries, but BPNPG7 and PNPG5 methods are limited to the clinical diagnostics field. It is important to demonstrate their relations to transfer these advanced methods to other fields.

The commercial a-amylase activities as determined by BPNPG7, are compared to those obtained using more traditional assay procedure: amylose azure, starch azure, Bernfeldmethods (Figure 10, 11, and 12). Assays were run 10 min at40°C for BPNPG7 and Bernfeld methods, 15 min at 60°C for Azure methods. Regression equations are listed for each comparison. The BPNPG7 a-amylase assay procedure correlates very well with the amylose azure, starch azure, and Bernfeld methods, with correlation coefficient 0.9937, 0.9958, and0.9993 respectively. The commercial E-amylase activities assayed by PNPG5 method is also compared to those obtained from Bernfeld assay procedure (Figure 13) and a high correlation coefficient 0.9920 was also found.

0b32

g 28 23 »> § s •ISf

c a

5

4

3

2

1

O0.1 0.2 0.3 0.4 0.5

a AMYLASE (uniVg on BPNPG7)

0.6

Figure 10. Relationship between the BPNPG7 and amylose azure method for measurement ofa-amylase. Correlation coefficient, r=0.994; Y = 1.32 + 5.94X.

a-A

MY

LA

SE

(u

nlt/9

on

star

oh

azu

re)

2.5

2.0

1.5

1.0

0 . 5

0 . 0 | ; 1 1 1 1--------O 0.1 0.2 0.3 0.4 0.5 0.6

a -A M Y L A S E (uniV9 on B P N F G 7 )

Figure 11. Relationship between the BPNPG7 and starch azure method for measurement ofa-amylase. Correlation coefficient, r=0.996; Y = 0.996 + 2.95X.

a-A

MY

LA

SE

2.5

2.0-eE0«o

1.5

! 1.0aEW 0.5

0.00.00 0.02 0.04 0.06 0.08 0.10 0.12

u -A M Y L A S E (untt/9 on BPNPG7)

Figure 12. Relationship between the BPNPG7 and soluble starch method (Bernfeld method)for measurement of a-amylase. Correlation coefficient, r=1.000; Y = -0.16 + 21.5X.

3-A

MY

LA

SE

(m

g m

alt

oae/

min

)

2.0

1.5

1.0

0.5

0.00.80.60.2 0.40.0

3-AMYlASE (uniVg on PNPG5)

Figure 13, Relationship between the PNPG5 and Bernfeld method for measurement of II-amylase. Correlation coefficient, r=0.992; Y = -0.025 + 2.03X.

88The sensitivity of an assay can be defined as the rate

of change of absorbance per unit of enzyme activity. The value is easy to obtain from the linear regression equation: Y * a + bX, where Y is absorbance and X is activity (Unit), a is constant, and b is sensitivity (AABS/per unit of enzyme activity). To make comparison among different assay methods, the same enzyme unit is used, BPNPG7 unit for a-amylase and PNPG5 unit for S-amylase. Table 6 shows the sensitivities of three a-amylase assay methods. BPNPG7 has the highest sensitivity (5.03), approximately 20 times more sensitive than the starch azure method and 10 times more than the amylose azure method. Since the a-amylase activity in sweetpotatoes is very low, the absorbance reading is only 0.110 at 595 nm for the raw juice (no dilution instead of four times dilution of standard extraction procedure) from 'Beauregard' by using amylose azure method at 60°C for 15 min. The BPNPG7 method shows great advantage on assaying sweetpotato a-amylase over amylose azure and starch azure methods.

Comparison of sensitivity between the two fi-amylase methods is shown in Table 7. Although the difference of sensitivity is not as great as that of a-amylase assay methods, the PNPG5 is still approximately two times more sensitive than the Bernfeld method.

89

Table 6. Comparison of sensitivity among three or-amylase assay methods.

Activity BPNPG7 Amylose azure Starch azureU/ml Absorbance1 Absorbance1 Absorbance1

0.0163 0. 0820 0.0292 0. 01420.0320 0.1610 0 . 0380 0.01800 . 0555 0.2790 0 .0460 0.02340 . 0787 0.3960 0 .0578 0.03060.0990 0.4980 0.0656 0 . 03280.1155 0 . 5810 0 . 0790 0.0378Sensitivity4: 5 .03 0 .475 0 .236AABS/1 U of BPNPG7

1 Absorbance (ABS) was determined at 410 nm in a spectrophotometer using standard BPNPG7 assay method.! Absorbance was determined at 595 nm in a spectrophotometer. Reaction condition was 2.7 ml of amylose azure reagent and 0.3 ml of various a-amylase preparations (prepared as described under Materials and Methods) at 60°C for 15 min and pH 6.0.1 Absorbance was determined at 595 nm in a spectrophotometer. Reaction condition was 2.7 ml of starch azure reagent and 0.3 ml of various a-amylase preparations (prepared as described under Materials and Methods) at 60°C for 15 min and pH 6.0.4 Sensitivity of an assay was defined as the rate of change of absorbance per unit of enzyme activity.

90

Table 7. Comparison of sensitivity between two 6-amylase assay methods: PNPG5 and Bernfeld methods.

6-AmylaseactivityU/ml

PNPG5Absorbance1

Bernfeld method Absorbance3

0.0123 0 . 064 0 .0090.0222 0 .115 0 . 0320.0262 0 .136 0 .0360.0493 0.256 0 . 0770.0754 0 .391 0 . 1170.1062 0.551 0 . 1570.1290 0 .669 0 . 2400.1550 0 . 804 0 . 288Sensitivity’ABS/l U of PNPG5

5 .18 1 . 89

1 Absorbance (ABS) was determined at 410 nm in aspectrophotometer using standard PNPG5 assay method.3 Absorbance was determined at 54 0 nm in a spectrophotometer using standard Bernfeld method.’ Sensitivity of an assay was defined as the rate of change of absorbance per unit of enzyme activity.

91D e t e r m i n a t i o n o f Km a n d V toax

The kinetic parameters (Km and Vmax) for hydrolysis of BPNPG7 were examined at 40°C for ex-amylases from four cultivars. The results obtained are presented in Table 8 calculated from Lineweaver-Burk plots (Figure 14).

Michaelis-Menten constants (Km) and substrate turnover numbers (Vmax) of sweetpotatoes were not the same among the four cultivars, with Centennial having the highest Vmax (0.722 U/g), Jewel having the lowest Vmax (0.406 U/g); little difference was observed between Beauregard and Hernandez, with 0.571 and 0.577 respectively. Centennial had slightly lower Km value (0.55 mM) than the other three cultivars, which indicates higher affinity and physiological efficiencies. No significant differences on Km values among the other three cultivars (from 0.62 to 0.65 mM) were found.

The kinetic parameters (Km and Vmax) for hydrolysis of PNPG5 were also examined at 40°C for S-amylases from the four cultivars. The results (calculated from Figure 15) are shown in Table 9. The Km and Vmax values of E-amylase from Centennial were significantly higher than those of the other three cultivars (1.73 mM). Jewel had a slightly higher value (0.91 mM) than Beauregard and Hernandez, which had similar values (0.81 and 0.82 mM).

No published information is available on kinetic parameters of sweetpotato amylases for hydrolysis of BPNPG7 and PNPG5. However, a Km of 0.607 mM BPNPG7 was previously

92

Table 8. Kinetic properties of sweetpotato ar-amylase for four cultivars for the hydrolysis of BPNPG7.1

Enzyme source Vmax (U/g) Km (mM)

BEAUREGARD 0 . 571 0 . 62CENTENNIAL 0 . 722 0 . 55HERNANDEZ 0 . 577 0 .65JEWEL 0 .406 0 .65

1 Reaction condition was 0.2 ml of various concentration of BPNPG7 and 0.1 ml of sweetpotato extract at 40°C for 10 min and pH 6.0.

O tO7 BEAUREG ARD

o CENTENNIAL8.0

A HERNANDEZ

6.0

4.0

2.0

-2.0 -1.0 0.0 1.0 2.0 3.0 4.0

1/S (1/mM)

Figure 14. Lineweaver-Burk plots for the hydrolysis of BPNPG7 by sweetpotato a- amylases from four cultivars. Linear regression equation for each cultivar is calculated as follows: Beauregard, Y = 1.75 + 1.09X; Centennial, Y = 1.78 + 0.76X; Hernandez, Y = 1.73 + 1.13X; and Jewel, Y = 2.46 + 1.61X.

V BEAUREGARD

O CENTENNIAL.0.0025 H

A HERNANDEZ

0.0020 H

0.0015 ~

o.ooio H0.0

1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5

1/S (1/mM)

Figure 15. Lineweaver-Burk plots for the hydrolysis of PNPG5 by sweetpotato fi-amylases from four cultivars. The linear regression equation for each cultivar is calculated as follows: Beauregard, Y = 0.000995 + 0.000809X; Centennial, Y = 0.000238 + 0.000412X; Hernandez, Y = 0.000958 + 0.000769X; and Jewel, Y = 0.000677 + 0.000615X.

95

Table 9. Kinetic properties of sweetpotato fi-amylase for four cultivars for the hydrolysis of PNPG5.1

ENZYME SOURCE Vmax (U/g) Km (mM)

BEAUREGARD 1004 . 0 0 . 82CENTENNIAL 4198 . 6 1 .73HERNANDEZ 1043 .7 0.81JEWEL 1474 . 4 0 .91

1 Reaction condition was 0.1 ml of various concentration of PNPG5 and 0.1 ml of sweetpotato extract at 40°C for 10 min and pH 6.0.

96reported for wheat a-amylase (McCleary and Sheehan, 1987) , which appears to be similar to sweetpotato a-amylase. The values of Km for a-amylases from other cereals were also reported {Sirou et al. 1990) . Rice a-amylase has a much lower Km (0.273 mM) than the others. Barley and maize a-amylases have a similar value (0.715 mM for maize, 0.730 mM for barley). Sorghum a-amylase Km is higher (0.992 mM). A Km of 0.32 mM PNPG5 was reported for 6-amylase from cereal (McCleary and Codd, 1989). Similar data were obtained for maize (0.37 mM) , rice (0.381 mM) and sorghum (0.386 mM) , and a slightly higher value for barley (0.466 mM) (Sirou et al. 1990) . The Km values obtained from sweetpotato 6-amylases (from 0.81 to 1.73 mM), were higher than for 6-amylases of the cereals, suggesting a lower affinity of sweetpotato 5- amylases.

Km is the substrate concentration at which the activity of the enzyme is half maximal. Maximal activity is only obtained at an infinite substrate concentration but, in practice, an enzyme is often considered saturated with substrate when [S] > 10 Km (when S = 10 Km, activity is infact 91% of maximal). Hence, sometimes term apparent Km (Km') is used instead of Km. Enzyme activities are normally reported at saturating substrate concentrations to facilitate comparisons because the reaction will approach zero order so that changes in substrate concentration will have no effect on the activity of the enzyme. From the Km values of a-

97amylase and 6-amylase in sweetpotato, a saturating BPNPG7 substrate concentration (lOKm) for a-amylase should be about 6 mmol, a saturating PNPG5 substrate concentration (lOKm) for 6-amylase should be between 8 and 17 mmol. Because a large number of assays were to be performed in this research, and a preliminary study showed that a lower substrate concentration for enzyme assay achieved satisfactory results, the saturating substrates were not employed in the enzymes assays for economy. Another reason is that the Km is relatively stable for enzymes from the same source. If Km is known, the Vmax of the enzyme can be calculated at any concentration of substrate by using the Lineweaver-Burk equation to compare the activities of enzyme assayed at a different concentration of substrate.Temperature effects on a - and ft-amylase assay

The enzymatic process is influenced the most by the temperature to which food is exposed during processing and storage. As temperature rises, the rates of chemical reaction increase, but high temperatures cause denaturation so enzymes lose activity. Figure 16 shows the effect of temperature on a- and 6-amylase assay in sweetpotatoes. The activity increased rapidly when temperatures increased from 3 0 to 4 0°C, and reach maximum at 40 and 50°C for a-amylase, and at 40°C for S-amylase. At 60 and 70°C, tremendous reduction in activity and smaller activity differences from four cultivars were observed. For 6-amylase assay, no differences were

98a-AMYLASE ASSAY

Eco

IIIo

EDB8 9

EEo

u0

BB89

BEAUREGARD

CENTENNIAL

HERNANDEZ

JEWEL

0.0

0.4

0.3

0.2

0 . 1

0.0

T E M P E R A T U R E C C )

P-AMYLA8E ASSAY

BEAUREGARD

CENTENNIAL

HERNANDEZ

JEWEL

T E M P E R A T U R E ( ° C )

Figure 16. Effect of temperature on BPNPG7 a-amylase assay and PNPG5 fl-amylase assay. Sweetpotato juice was extracted from Beauregard and assayed using standard procedure except using temperature 30, 40, 50, 60, and 7 0°C for incubation respectively.

99observed among different cultivars at these temperatures. It appears that BPNPG7 and PNPG5 assay methods are not suitable for sweetpotato a- and E-amylase assay at high temperatures (60 and 70°C) due to the denaturation of enzymes. Since assay reagent contained a-amyloglucosidase, decrease of activity could be due to denaturation of native enzymes in sweetpotatoes, as well as a-amyloglucosidase. The information about thermal stability of a-amyloglucosidase is not available from the supplier, Sigma Chemical Company. To have a better understanding of the effects of both native and commercial enzymes, there is a need to conduct further study on the thermal stability of a-amyloglucosidase in addition to native enzymes.Preparation of a-Amylase Reagent

The a-amylase reagent was a commercially available product from Sigma normally used to assay a-amylase in human serum and urine. Information on its application in other a- amylase assays is not available. The reagent consists of BPNPG7, a yeast a-glucosidase, phosphate buffer, and stabilizers, which are essentially the same as those included in the substrate mixture for a-amylase assay as described by McCleary and Sheehan (1987) . Also, it is much more convenient and cheaper to buy this ready-to-use reagent than to prepare a mixture by oneself. Therefore, this reagent was used in sweetpotato a-amylase assay with modification. To change the pH from its normal value of 7.0 to 6.0, the reagent was

100reconstituted in pH 6.0 phosphate buffer rather than in distilled water.Preparation of fi-Amylasa Raagant

The fi-amylase substrate mixture used in this study contained PNPG5 (5 mM) , a-glucosidase {100 U/ml) and was prepared in distilled water (McCleary and Codd, 1989) . However, the substrate was not as stable as described by others. It became unusable within 24 hrs storage at 4°C, instead of 6 days. This may have been due to contamination of amylases in the a-glucosidase. Therefore, it was necessary to prepare the substrate mixture fresh just before assay. Calculation of Enzymt Activity

Enzyme activity is usually reported in term of the amount of product formed per unit time with the standard unit ^imol/min. For assays on crude homogenates it is necessary to relate the activity to the amount of tissue. The simplest course is to report data in terms of wet weight of the tissue, e.g., |tmol/min/g wet weight. Enzyme activity may be reported in terms of dry weight to eliminate any influence of change in water contents under different conditions. This may be particularly important when testing different plant tissues. Because of this, activities are frequently expressed in terms of protein content. Enzyme activities in tissues are frequently rendered incomparable by the use of different reference points. This can be overcome by providing a variety of information; for example, wet weights and protein content.

101The apectrophotometrie assay is probably the most widely

used of all assay methods, which are based on a directproportionality between absorbance and concentration (Beer's Law) . Each absorbing species can be characterized by its molar extinction coefficient, E* which is the absorbance, at a specified wavelength and fixed path-length (usually l cm) , of the species when present in the light path at aconcentration of 1 mol/L (or mmol/ml). Therefore, if the absorbance of a solution is A, the volume of the solution in the cuvette is V, the amount of absorbing species present will be equal to (V.A/EJ mmol or (V.A.10VEJ /imol. Thisequation can be easily used to calculate the activity of sweetpotato amylases as follows:Sweetpotato amylase activity (U/g fresh sample) =

A^ 4io total vol. 1 extraction.x x __,___ xdilutionincubation time aliquot assayed E^ volume

To see how to calculate enzyme activity from this equation, an example of the 6-amylase assay is as follows: incubation time = 10 min, total volume in cell = 3.2 ml, aliquot assayed = 0.1 ml, E„ of p-nitrophenol in 1% Trizma base = 16.6,extraction volume = 4 ml/g fresh sample, dilution = 4000, the calculated activity would be:

units/g = x 3 ‘ x ------- - x x 400010m m 0.2ml 16.6ml/jimol g

= AA,i„ x 3084.3

102E„ of p-nitrophenol in IV Trizma base was calculated

from the linear equation of standard curve of p-nitrophenol: Y « a + bX, where Y is absorbance, X is concentration (jxmol/ml) , then b will be the E„. It was previously reported that Em of p-nitrophenol is 18.8 (Sirou et al. 1990) and 17.0 (McCleary and Codd, 1989). The data obtained in this research is slightly lower with 16.6 ml/jmol by using the p- nitrophenol standard kit from Sigma (Catalog No. 140-1) . The difference may due to the differences in instruments and purity of p-nitrophenol standards.

To convert unit/g fresh weight to unit/g dry weight and unit/mg protein, dry matter content and protein content need to be measured. The unit/g dry matter and unit/mg protein can be easily obtained by dividing unit/g fresh weight by dry matter and protein content.EXPERIMENT 2. CHARACTERIZATION OF SOME PROPERTIES OF SWEETPOTATO AMYLASESTemperature Effect on Stability of Amylaaea

It is well known that the amount of final products produced during enzymatic hydrolysis not only depends on the enzyme activity but also on enzyme stability. Previously, the stabilities of sweetpotato amylases were investigated under special circumstances, e.g., purification, added buffer with stabilizer. However, most sweetpotato processing is conducted in natural occurring conditions, like baking, without purifying enzyme or adding buffer with stabilizer. Therefore, to provide the sweetpotato industry more realistic

103information relative to amylase stability, it is necessary to investigate the properties of the enzymes in crude preparations.

To determine the effect of temperature on amylase stability, 'Beauregard' sweetpotato juice was prepared as with the standard extraction procedure except that distilled water was used instead of buffer for enzyme extraction.

The effects of temperature on a-amylase stability are shown in Figure 17. The enzyme activity was quite stable at -20°C; after four weeks of storage, no activity loss was detected. At 4°C, a gradual decline in activity was observed, with a drop to a value of 70% of original level after 4 weeks of storage. At room temperature storage, the activity was completely lost within 6 days. The loss of activity in a shorter time at room temperature could be due to faster growth of microorganisms, which could inhibit or destroy enzyme activity. As expected, an increase in temperature inactivated a-amylase more quickly. At 75°C, the loss of activity was very rapid initially, decreasing to 40% of the original value in just 30 seconds of heating, then a slower decline to 13% of the original value after 5 min heating. Thus, it appears that the stability of the a-amylase is temperature-dependent. However, S-amylase is much more stable than a-amylase as shown in Figure 18; no significant activity change occurred during 4 weeks storage at -20, 4, and 25°C. The stability of sweetpotato E-amylase at 4 and -23°C was

104

w 10O-

80

60-

TIME (minutes)

£

S§III>5ina

80

60

20 CRT

2 0

20 25 30lO 15

TIME (days)

Figure 17. Stability of a-amylase in sweetpotatoes at different temperatures. Sweetpotato juice was extracted from Beauregard by distilled water. Activity was expressed relative to that of fresh samples immediately after extraction and determined by standard BPNPG7 procedure.

105

TIME (minutes)

1 O O -

>sg3uiDC

80-

60-

20°C2 0

lO 30

TIME (minutes)

Figure 18. Stability of fl-amylase in sweetpotatoes at different temperatures. Sweetpotato juice was extracted from Beauregard by distilled water. Activity was expressed relative to that of fresh samples immediately after extraction and determined by standard PNPG5 procedure.

106reported by Hiranpradit and Lopez (1976) . They also found no significant change in activity within 28 days storage period. At 75°C, the loss of activity is also slower than with a- amylase, decreasing to only 60V of original level after 10 minutes heating, compared to 40% of original level within 3 0 seconds for a-amylase.

The results obtained in this study show IS-amylase is much more stable than a-amylase, which is quite different from previous research. It was reported that a-amylase is heat stable with an optimum temperature from 70° to 75°C (Ikemiya and Deobald, 1966). Most activity remained after one hour of heating and there was a 60% loss of the original value after three hours of heating at 70°C. This difference may be due to the use of calcium chloride as a stabilizer for enzyme preparation in the previous study. Another possible reason could be the different assay temperature used for the a-amylase determination. A high incubation temperature of 70°C was used for a-amylase assay previously. At this temperature, most activity could be lost very quickly and only remaining activity was actually measured. Therefore, the great decrease of activity occurring in the early stage could be no longer detected. The results obtained could only demonstrate the change of remaining activity, which is relatively stable after 30 seconds heating at 75°C as shown before.

107Conpariaon of tho Stability of Anylaaaa in Diffarant Cultivara.

Figure 19 shows the stability of a-amylase in four different sweetpotato cultivars at 75®C. 'Jewel' had the lowest stability. Its activity decreased to 13% of the original value after 30 seconds of heating and was fully inactivated in 5 min. No significant differences existed among the other three cultivars. For all cultivars, most activity was lost within 30 seconds, then gradually declined to below 15% of the original value after 5 min of heating.

The stability of 6-amylase in the four sweetpotato cultivars at 75°C was also examined as shown in Figure 20. 'Centennial' had the highest stability, more than 3 0% of the original activity still remained after 30 minutes of heating time. There were no significant differences among the other three cultivars. For all cultivars, 6-amylase contrasted with a-amylase where its activities decreased very rapidly at the beginning, then changed very slowly at late stage. The activities of 6-amylase decreased gradually during the whole heating period. Most activity remained after 10 minutes of heating. A similar pattern was reported by Nakayama and Kono (1957) in a sweetpotato 6-amylase heat inactivation study. The experiment was conducted at 63°C and showed that most activity remained after 20 min of heating.

It appears that there is a relationship between enzyme activity and stability. For example, 'Jewel' had the lowest a-amylase activity, and also had the lowest stability in a-

100.0 BEAUREGARD

CENTENNIAL

HERNANDEZ

JEWELBO.O

TIME (minutes)

Figure 19. Stability of a-amylase from four different sweetpotato cultivars at 75°C. Sweetpotato juice was extracted from four cultivars respectively by distilled water. Activity was expressed relative to that of fresh samples immediately after extraction and determined by standard BPNPG7 procedure.

100.0 BEAUREGARD

CENTENNIAL

HERNANDEZ

JEWEL80.0

60.0

40.0

TIME (minutes)

Figure 20. Stability of A-amylase from four different sweetpotato cultivars at 75°C. Sweetpotato juice was extracted from four cultivars respectively by distilled water. Activity was expressed relative to that of fresh samples immediately after extraction and determined by standard PNPG5 procedure.

110amylase; 'Centennial' had the highest S-amylase, and also had the highest stability in S-amylase. However, enzyme activity is not the only factor that affects the enzyme stability. From the dilution effect experiment, after an 8 times dilution of 'Beauregard' juice, the Of-amylase activity became lower than that of 'Jewel' juice obtained from the standard extraction procedure, yet the stability of a-amylase in 'Beauregard' was still much higher than that in 'Jewel'. Therefore, the native property of enzyme is another important factor that affects enzyme stability.Dilution Effect on the Stability of Amylases

Since the 8 times dilution treatment was used in this study, and the a-amylase level in sweetpotatoes was low, the standard extraction procedure {4 times dilution) for sweetpotato juice could cause low readings of absorbance after further dilution. Therefore, the juice was obtained by squeezing the grated roots in a Shear press without dilution, and then centrifuging at 13, 200G for 10 min (4°C) . The supernatant collected was used as 1 times dilution treatment. The dilutions of 4 times, and 8 times were prepared by adding 3 and 7 volumes of distilled water to 1 volume of the juice.

The effect of dilution on stability of a-amylase can be seen in figure 21. No statistical differences were seen among the three treatments of 1, 4, and 8 times dilution. However, at the end of 5 min reaction time, the 8 times dilution treatment had lower remaining activity than those of 1 times

#w

>

I

III>5uE

100.0

80.0

60.0

40.0

20.0

0.0

IX DILUTION

4X DILUTION

8X DILUTION

TIME (minutes)

Figure 21. Effect of dilution of sweetpotato a-amylase on its heat stability at 75°C, Sweetpotato juice was squeezed from grated Beauregard roots and used as IX dilution. Dilutions of 4X and 8X were made by adding distilled water. Activity was expressed relative to that of fresh samples immediately after preparation and determined by standard BPNPG7 procedure.

112and 4 times dilution treatments. The small difference among the 3 treatments may be due to insufficient dilution of juice. For heat stable commercial a-amylase from BacilluB licheniforms, a 5,000 times dilution is needed to observe a significant effect of dilution on thermal stability (Table 10) .

The effect of dilution on stability of S-amylase is more sensitive than that of a-amylase as shown in Figure 22. The 8 times treatment had significantly lower stability than the 4 times and 1 times dilution treatments. The increased difference between 4 times and 1 times treatment can also be seen as the time of heating increased. It was reported that B-amylase of sweetpotato is a terameric enzyme with a molecular weight of 215,000 and consists of four identical subunits. The enzyme dissociates into monomers with no activity at high dilution {Bernfeld et al. 1965). This could be the reason that the 8 times treatment had a greater effect on S-amylase than on a-amylase.Interaction of Commercial a-Amylaee and ft-Amylase on Starch Hydrolyaie

It was found that the combined action of a-amylase and S-amylase was more effective than the action of a-amylase or B-amylase alone (Maeda et al. 1978). Similar results were observed in this research when high concentrations of amylases (higher than normal assay concentration) were incubated with 1% gelatinized sweetpotato starch at 40°C for 10 minutes. A mixture was prepared by mixing equal volumes of

Table 10. Effect of dilution on the stability of commercial or-amylase.1

Treatment Time (min) 0 5 10 20 50500X Activity (U/ml)1 0.908 0. 930 0. 920 0 . 918 0.896

% Activity1 100.0 102 .4 101.3 101.1 98.75000X Activity (U/ml) 0.091 0.078 0.018

\ Activity 100.0 85.3 20.1

1 Commercial alpha-amylase used here is Bacillus licheniform from International.1 Enzyme activity was measured at 410 nm in a spectrophotometer using standard assay method.1 Enzyme activity is expressed relative to that of unheated controls.

QuestBPNPG7

*w

15>P8Id>5uE

100.0

80.0

60.0

40.0

20.0

0.0

1X DILUTION

^ 4X DILUTION

8X DILUTION

10 15 20

TIME (minutes)

25 30

Figure 22. Effect of dilution of sweetpotato B-amylase on its heat stability at 75°C. Sweetpotato juice was extracted from Beauregard following standard procedure by distilled water and used as IX dilution. Dilutions of 4X and 8X were made by adding distilled water. Activity was expressed relative to that of fresh samples immediately after preparation and determined by standard PNPG5 procedure.

114

115a-amylase and S-amylase. The total amylase activity in the mixture should be the same as the average value of those of a-amylase and S-amylase if no synergism existed. Therefore, the effect of interaction can be compared on the same activity basis. Figure 23 shows the synergistic interaction of a-amylase and E-amylase at high concentrations of amylases. The maltose produced by the mixture can be as high as 50% more than the average value. However, the effect became smaller as the enzyme concentration decreased. Finally, at normal assay concentration (where concentration is directly proportional to maltose produced), no significant differences were observed (Figure 24) . When the concentration of enzyme was further decreased, the maltose produced by the mixture was even lower than average value.

Therefore, the synergistic hydrolysis of granules could occur when S-amylase is combined with a-amylase, but it is not always true, depending on the concentration of amylase. This behavior may be due to following reasons. First, at normal enzyme concentrations, excess of substrate is available for both a-amylase and E-amylase, and the limiting factor for starch hydrolysis is enzyme concentrations, which is proportional to maltose produced. Since the enzyme concentrations in the mixture are equal to the average concentration of a- and S-amylases alone, the maltose produced should also be the same. Secondly, at high concentrations of amylases, the limiting factor changes from

8.0

E 6.0-

4.0

MIXTURE

AVERAGE

0.00.0 20.0 60.0 80.0 100.0

CONCENTRATION (% of the dilution)

Figure 23. Interaction between a- and A-amylase on sweetpotato starch hydrolysis at high enzyme concentration. A. 1/10,000 dilution of commercial a-amylase and 1/1,000 of dilution of commercial B-amylase were further diluted by 100, 80, 60, 40, and 10% of these concentrations. The values obtained from the mixture of a- and A-amylase were compared with the average value obtained from a- and A-amylase alone.

116

2.5

| 2.0

NaE 1.5wUlw0 i.oHJ<g 0.5

0.0 ------ J-- !----- j----- j----- j----- 1----- ]----- [----- 1----0.0 20.0 40.0 GO.O 80.0 100.0

CONCENTRATION (% of the dilution)

Figure 24. Interaction between a- and fi-amylase on sweetpotato starch hydrolysis at normal enzyme concentration. A 1/100,000 dilution of commercial a-amylase and 1/10,000 dilution of commercial B-amylase were further diluted by 100, 80, 60, 40, and 10% of these concentrations. The values obtained from the mixture of a- and B-amylase were compared with the average value obtained from a- and 8-amylase alone.

118enzyme concentration to substrate availability. The S-amylase cleaves a-(1-4) D-glucosidic linkages in starch components in a stepwise fashion from the nonreducing end. The a-amylase hydrolyzes interior a-(1-4) linkages of starch randomly. Every time it breaks down an interior linkage, a nonreducing end will be produced, hence a-amylase could increase the substrate availability for the hydrolysis of S-amylase. Especially when the action of S-amylase stops in the region of a-(1-6) linkages, a-amylase can break down the linkage after the branch point and allow E-amylase to continue further hydrolysis. On the other hand, a-amylase can also benefit from S-amylase because S-amylase attacks short chains faster than a-amylase does. Since S-amylase of sweetpotato is a terameric enzyme and dissociates into monomers with no activity at high dilutions (Bernfeld et al. 1965), when the concentration is very low, this high dilution could reduce enzyme stability in the mixture. The actual activity of a- amylase and S-amylase is only half of that of a-amylase or of E-amylase alone, and the mixture produces less maltose than the average because of reduced stability of the enzyme in the mixture.

Another hypothesis for the synergistic interaction between a-amylase and S-amylase was suggested by Maeda et al. (1978) . S-Amylase was thought to spare the a-amylase activity by digesting the dextrin solubilized by the action of a- amylase on starch granules. These in turn are digested

119further by the a-amylase itself in the absence of added £-amylase. Thus, in the presence of added S-amylase, a-amylaseactivity may be focused on the digestion of starch granulesrather than to the digestion of solubilized dextrin, its owndigestion products. The reason that a-amylase can increasethe hydrolysis of the S-amylase was not explained.EXPERIMENT 3, CHANGES OF AMYLASE ACTIVITIES AND CARBOHYDRATE CONTENTS IN SWEETPOTATOES DURING STORAGE AND THEIR EFFECTS ON VISCOSITY OF SWEETPOTATO PUREEThe Effect of Storage on the Activities of a- and fi-Amylase in Four Sweetpotato Cultivars.

The a-amylase activity expressed as unit per g fresh weight (Figure 25 and Table 11) showed differences (PsO.Ol) among cultivars during a four month storage period except between cultivars 'Beauregard' and 'Hernandez'. 'Centennial' had the highest a-amylase activity, while 'Jewel' had the lowest (only about half as much as that in 'Centennial') . No published information is available for amylase activity in 'Beauregard' and 'Hernandez' cultivars. It was reported that 'Centennial' had a higher a-amylase activity than that in 'Jewel' (Walter et al. 1975). However, analyses of the a- amylase activity within cultivar (Figure 25 and Table 11) showed that during storage little change took place over a period of four months after harvest. Although a slight increase (about 20%) was observed in 'Centennial', 'Beauregard', and 'Hernandez' cultivars after four months of storage, the results obtained in this study are very different from those reported previously. Walter et al.

0.5

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c3w

BEAUREGARD

CENTENNIALO.H HERNANDEZ

JEWEL0.0

C 2 3 AH 1

STORAGE TIME (months}

Figure 25. a-Amylase activity for four sweetpotato cultivars at harvest (H}, after curing {C), and during four months of storage. Activity was expressed as unit/g fresh weight on BPNPG7 substrate*

120

121

Table 11. Effect of storage time on a-amylase activity infour sweetpotato cultivars.

a-Amylase activity (U/g fresh wt.)

Storagetime(month)

CultivarsBeauregard Centennial Hernandez Jewel

AT HARVEST 0 .296 0 .338 0 . 263 0 .233AFTER CURING 0 . 287 0 . 366 0 .270 0 . 2421 MONTH 0 . 282 0.332 0 .256 0 . 1992 MONTHS 0 . 292 0.408 0.293 0 .2133 MONTHS 0 . 341 0.421 0 .293 0 .2144 MONTHS 0 .318 0 .417 0 . 309 0 .215

Significance NS1 NS NS NS

Mean 0.303 BJ 0 . 380 A 0.281 B 0 .219 C

1 Means separation by Duncan's multiple range test. NS = No significant difference within columns at P s 0.05.’ Means separation by Duncan's multiple range test. Means within a row followed by the same letter are not significant at P £ 0.05.

122(1975) found that a 40-fold increase of a-amylase activity occurred in 'Jewel' and 'Centennial' in 71 days of storage. Morrison et al. (1993) reported that a-amylase activity in 'Jewel' rose after harvest and reached a maximum (about 25- fold increase) after 90 days, then decreased by 50% until the last sampling date (180 days). Only the results reported by (Ikemiya and Deobald, 1966) were similar to the present study. It was found that a small increase of a-amylase activity in 'Goldrush' cultivar occurred in the early stage of storage, from 3.4 0 SDU/ml at harvest to 3.85 SDU/ml after 42 days of storage. About a two-fold increase occurred after 95 days of storage. It is difficult to explain the differences in results because of the many factors that may be involved. It seems that different assay methods used by different workers could be one of the main reasons. Four groups used three different assay methods, and of those two groups using the same method used very different enzyme juice preparation procedures.

The S-amylase activity expressed as unit per g fresh weight (Figure 26 and Table 12) also showed significant differences (PsO.Ol) among cultivars during a four month storage period, except between cultivars 'Beauregard' and 'Hernandez'. 'Centennial' was highest in S-amylase activity, 1.5 times higher than 'Jewel' and 2.5 times higher than 'Beauregard' and 'Hernandez'. Interestingly, the protein contents in the four cultivars displayed a similar pattern

[3-A

MY

LA

SE

AC

TIV

ITY

(un It

/g fre

sh wt)

3000.0

2500.0

2000.0

1500.0

1 0 0 0 . 0

500.0

0.0 ~f i i i i , i r~ t

H C 1 2 3 a

STORAGE TIME (months)

B E A U R E G A R D

CENTENNIAL

HERNANDEZ

JEWEL

Figure 26. fl-Amylase activity for four sweetpotato cultivars at harvest (H), after curing (C), and during four months of storage. Activity was expressed as unit/g fresh weight on PNPG5 substrate.

124

Table 12. Effect of storage time on fi-amylase activity in four sweetpotato cultivars.

6-Amylase activity (U/g fresh wt.)

Storagetime(month)


AT HARVEST 644 2060 582 756AFTER CURING 618 2212 569 7841 MONTH 614 2008 631 8042 MONTHS 612 2343 682 8473 MONTHS 704 2438 771 9634 MONTHS 632 2201 691 950

Significance NS1 NS NS NS

MEAN 637 C2 2210 A 637 C 850 B

1 Means separation by Duncan's multiple range test. NS = No significant difference within columns at P s 0.05.1 Means separation by Duncan's multiple range test. Means within a row followed by the same letter are not significant at P s 0.05.

125(Table 13) . The average protein contents in ' Centennial' during four months of storage was 1.3-fold higher than 'Jewel' and 2.6-fold higher than 'Beauregard' and 'Hernandez'. These results suggest that most of the protein in sweetpotatoes could be B-amylase protein. Since specific activity is expressed as U/mg protein, which can be calculated by dividing activity (U/g fresh wt,) by protein content (mg/g fresh wt.). Therefore, specific activities of E-amylase among four cultivars should have no significant difference.

Similar to Of-amylase, for all four cultivars no significant change (P>0.05) occurred over a period of four months of storage after harvest. However, a slight increase can be observed in some cultivars (e.g. 'Jewel'). The results obtained here are similar to those reported by Walter et al. (1975), where it was found that no significant increase occurred in S-amylase activity during 71 days of storage in both 'Jewel' and 'Centennial'. The activity in 'Centennial' was approximately two times higher than 'Jewel'. However, different results were obtained by Morrison et al. (1993) . Itwas reported that S-amylase in 'Jewel' cultivar increased by about 20-fold after 90 days of storage then decreased, then increased again until the last sampling date (180 days). The reasons for this difference are still unknown.

The a- and S-amylase activity in 'Jewel' harvested in 1993 was also investigated using BPNPG7 and PNPG5 methods.

126

Table 13 . Comparison of protein content in four sweetpotato cultivars.

Protein content (mg/lOOg fresh wt.)

Storagetime{month)


AT HARVEST 52 . 8 138 . 1 44 . 3 58 . 4AFTER CURING 37 . 1 113 . 2 29 . 7 42 . 91 MONTH 34 . 1 113 . 0 32 . 9 52 .82 MONTHS 19 . 0 113 . 2 30 . 1 52 . 03 MONTHS 28 .4 120. 9 39 . 5 54 . 54 MONTHS 25 . 2 113 . 2 28 . 2 55 . 8

Mean 32.75 C1 118.6 A 34.13 C 52.73 B

1 Means separation by Duncan's multiple range test. Means within a row followed by the same letter are not significant at P s 0.05.

127All assay samples were prepared according to standard procedure except that they were kept at -20CC for at least ten months before assay. Surprisingly, the results obtained from 1993 crop were very close to those from the 1994 crop (Figure 27) . It appears that amylase activities in 'Jewel' do not differ much from year to year. It also appears that frozen samples at -20°C could be an appropriate way to preserve amylase activity for a long period.Carbohydrate Changes during Storage and Puree Processing Reducing sugars

Glucose and fructose were the only reducing sugars detected by HPLC in raw roots. However, the reducing sugar contents in some cultivars (e.g. 'Centennial') were so low,that it was difficult to detect small changes that occur in these cultivars during storage. Therefore, the more sensitive colormetric method was used in this study to determine reducing sugar. Those sugars with higher concentration (sucrose and maltose) were still determined by HPLC. Storage generally increased the reducing sugar content in sweetpotatoes (Figure 28 and Table 14) , with most of the increase occurring during the curing period except for 'Centennial' (about 2-fold increase for 'Beauregard' and 'Hernandez', 5-fold increase for 'Jewel'). After curing, 'Jewel' continued to increase at a slower pace, but little increase occurred in 'Beauregard' and 'Hernandez'. The reducing sugar contents in the 'Centennial' cultivar

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Figure 27. Comparison of a- and B-amylase activity of Jewel cultivar sweetpotatoes harvested in different years at harvest (H), after curing (C), and during four or six months of storage. Fresh samples were used for amylase activities assay in the sweetpotatoes harvested in 1994, frozen samples were used for activity assay for the sweetpotatoes harvested in 1993.

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Figure 28. Reducing sugar {glucose and fructose} contents in four sweetpotatocultivars at harvest (H), after curing {C), and during four months of storage.

129

130

Table 14. Effect of storage time on reducing sugars(glucose + fructose) in raw sweetpotato roots.

Reducing sugar (g/100 g fresh wt.)

Storage Cultivarstime ----------------------------------------(month) Beauregard Centennial Hernandez Jewel

AT HARVEST 0 .59 a* 0 .17 a 0 . 73 a 0 .20 aAFTER CURING 1 .63 b 0.22 ab 1 . 54 be 0 . 99 b1 MONTH 1.51 b 0.27 b 1 . 67 be 1.06 b2 MONTHS 1 . 55 b 0.41 c 1 . 76 be 1.19 b3 MONTHS 1 .47 b 0.45 cd 1. 79 c 1 . 26 b4 MONTHS 1 .60 b 0.50 d 1.43 b 1 . 34 b

1 Means separation by Duncan's multiple range test. Meanswithin columns followed by the same letter are notsignificant at P s 0.05.

131increased gradually during the four months of storage, from 0.17% to 0.50%. 'Beauregard' and 'Hernandez' had the highest reducing sugar contents, while 'Centennial' had the lowest. It was suggested that source of glucose and fructose in sweetpotatoes during storage was probably from starch degradation and not sucrose (Picha, 1987).

During puree processing, little change took place in glucose and fructose contents {data not shown), which is consistent with reports by Walter and Hoover (1984), and Deobald et al. (1969). However, a large amount of another reducing sugar, maltose, was produced {Figure 29 and Table15) due to enzymatic conversion of starch. About 10.5% maltose (based on fresh weight), which is equal to 40% of AIS, formed in the puree processed from 'Centennial' and 'Jewel' cultivars at harvest, which decreased to 8.5% for 'Centennial' and 7% for 'Jewel' after four months of storage. Only 7.5% maltose was produced in the puree processed from 'Beauregard' and 'Hernandez' at harvest, which decreased to about 6% after four months of storage. The storage times and cultivars significantly affected the amount of maltose produced in puree processing, but not the ratios of maltose produced to AIS in raw roots (Table 16) . The ratio is approximately 40% and nearly consistent for all cultivars over the four months of storage. It seems that the amount of maltose produced during processing is AIS dependent and could be predicted from AIS content in raw roots.

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Figure 29. The amount of maltose produced (based on fresh weight) in processedsweetpotato puree from four sweetpotato cultivars at harvest (H), after curing (C),and during four months of storage.

132

133

Table 15. Effect of storage time on maltose producedduring sweetpotato puree processing.

Maltose (g/100 g fresh wt.)

Storage CultivarBtime ----------------------------------------(month) Beauregard Centennial Hernandez Jewel

AT HARVEST 7 .66 ab 10 .65 a 7 . 75 a 10.28 aAFTER CURING 8 .30 a 9.64 a 7 . 01 ab 8.59 be1 MONTH 8 . 07 a 8.51 a 7 . 56 a 8.18 cd2 MONTHS 7 . 07 be 9.35 a 7.47 a 8 . 89 be3 MONTHS 6 .30 cd 9.03 a 6 . 52 ab 7.63 cd4 MONTHS 6 . 05 d 8 . 54 a 5 . 77 b 7.14 d


Table 16. Effect of storage time on the ratios of maltose produced during processing and AIS in raw roots, the ratios of AIS change during processing and AIS in raw roots.

Ratio of maltose produced during processing and AIS in raw roots (%)

Harvest Curing 1 Month 2 Month 3 Month 4 Month Average

BEAUREGARD 40.47 47.27 47. 90 44.43 41.15 41.31 NS1 43.75CENTENNIAL 40.75 39.63 38.02 40.85 40.58 39.29 NS 39.85HERNANDEZ 38.56 42.49 46.71 46.61 42.90 39.06 NS 42.72JEWEL 41.65 39.00 37. 93 41.91 37.11 37.31 NS 39.15

Ratio of AIS change during processing and AIS in raw roots (%)

Harvest Curing 1 Month 2 Month 3 Month 4 Month AverageBEAUREGARD 44 .13 48.30 51.55 48.19 48.48 48.70 NS 48.22CENTENNIAL 41.43 43 .73 45.04 48.44 43.66 43.81 NS 44 .35HERNANDEZ 47. 14 47.59 50.18 46 .14 47.55 48.94 NS 47. 93JEWEL 40.28 45.42 47.75 45.60 44.52 45.43 NS 44 .83

1 Means separation by Duncan's multiple range test. NS = No significant difference within columns at P s 0.05.

135Sucrose

Sucrose contents were approximately 2% (based on fresh weight) for all four cultivars at harvest (Figure 30 and Table 17) . An increase in sucrose content occurred in 'Centennial' and 'Jewel' cultivars during curing, with a more rapid increase in 'Centennial'. However, only a small amount of sucrose increase was observed in ' Beauregard' and 'Hernandez' cultivars during curing. After curing, little increase occurred for all cultivars as length of storage increased. A possible metabolic pattern of sucrose formation was suggested by Picha (1987) that the substrate for sucrose probably was starch.

No significant change occurred in sucrose during puree processing (data not shown), which confirmed previous results (Walter and Hoover, 1984, Deobald et al. 1969).Total sugar

Total sugar contents in 'Beauregard' and 'Hernandez' were slightly higher than those in 'Centennial' and 'Jewel' at harvest, but after four months of storage, higher values were observed in 'Centennial* and 'Jewel' (Figure 31 and Table 18). An increase of total sugar contents occurred during curing for all four cultivars. However, after curing, no significant increase occurred as length of storage increased except in 'Jewel', which increased from 4% to 5%.

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HERNANDEZ

■*- JEWELo a o 0■"y9w 3.0 hito0E 2.0 0 310

1.0H C 1 2 3 A


Figure 30. Sucrose contents in four sweetpotato cultivars at harvest (H) , after curing(C), and during four months of storage.

136

137

Table 17. Effect of storage time on sucrose content inraw sweetpotato roots.

Sucrose (g/100 g fresh wt.)


AT HARVEST 2 .18 a1 2 . 07 a 1. 94 a 2 . 07 aAFTER CURING 2 .47 ab 3 .80 b 2 . 17 ab 3 . 05 b1 MONTH 2 . 26 ab 3 . 82 b 2 .29 b 3 . 07 b2 MONTHS 2 . 24 ab 3 .01 b 2 . 37 b 3 .17 be3 MONTHS 2 .40 ab 3 . 52 b 2 .27 b 3 . 05 b4 MONTHS 2 . 56 b 3.81 b 2 . 30 b 3 . 57 c


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STORAGE TIIME (months)

Figure 31. Total sugar contents in four sweetpotato cultivars at harvest (H), aftercuring {C), and during four months of storage.

138

139

Table 18. Effect of storage time on total sugar contentin raw sweetpotato roots.

Total sugar (g/100 g fresh wt.)


AT HARVEST 2 . 77 a1 2 .24 a 2 .68 a 2 . 27 aAFTER CURING 4 . 10 b 4 . 02 b 3 . 71 b 4 . 04 b1 MONTH 3 .76 b 4 .09 b 3 . 96 b 4 . 14 b2 MONTHS 3 . 79 b 4.41 b 4 . 14 b 4 .35 be3 MONTHS 3 . 88 b 3 . 97 b 4 . 06 b 4 .31 be4 MONTHS 4 . 15 b 4.31 b 3 . 73 b 4 . 91 c


140Alcohol insoluble solids (AIS)

For all four cultivars, as length of storage increased, AIS content decreased {Figure 32 and Table 19). The largest decrease occurred in the first month of storage. 'Centennial' had the highest AIS content; 'Beauregard' and 'Hernandez' the lowest. AIS contains mostly starch. It is generally believed that the decrease of AIS during storage is due to starch being converted to reducing sugars and sucrose. However, it was noticed that the amounts of AIS change were not equal to the amounts of total sugar change (Table 20). After 4 months storage, the change of total sugar was less than 50% of AIS change. For example, the AIS decreased by 4.82 g/lOOg fresh wt. in 'Beauregard', but total sugar only increased by 1.38 g/lOOg fresh wt. It seems that starch conversion into sugar cannot completely explain all changes in AIS. Later, it was noticed that dry matter in all four cultivars decreased significantly (P<0.01) as length of storage increased {Figure 33). After four months of storage, 'Jewel' decreased to the greatest extent by 3.63% (g/lOOg fresh wt.) compared to the amount at harvest; 'Centennial' had the least change with 1.75%. 'Beauregard' and 'Hernandez' decreased by 2.63% and 2.34% respectively. Obviously, dry matter decrease is not because of moisture loss, which can only increase dry matter content. Therefore, the loss in dry matter is possibly caused by respiration, which converts starch in AIS into C0a and Hs0 and reduces dry matter contents. Further, comparing the AIS

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Figure 32. Alcohol-insoluble solids (AIS) content of four sweetpotato cultivars atharvest (H), after curing (C), and during four months of storage.

141

142

Table 19. Effect of storage time on AIS of rawsweetpotato roots.

AIS (g/100 g fresh wt.)


AT HARVEST 19.47 a1 26 . 20 a 20 .23 cl 24.67 aAFTER CURING 17 . 64 b 24 .46 ab 16 .59 b 21.99 b1 MONTH 16 .87 be 22 .39 ab 16 . 22 b 21.54 b2 MONTHS 15 . 91 be 22 . 79 ab 16 .09 b 21.16 be3 MONTHS 15.40 be 22 .35 ab 15 . 23 b 20.65 be4 MONTHS 14 . 65 c 21 .69 b 14 . 82 b 19.07 c


143

Table 20. Change in AIS, total sugar and dry matter during storage compared to the amounts at harvest.

Change in total sugar during storage (g/lOOg fresh wt)

Months Curing 1 2 3 4BEAUREGARD 1.33* 0 .99 1 . 02 1. 10 1 . 38CENTENNIAL 1.79 1 . 85 2 . 18 1.73 2 .07HERNANDEZ 1. 03 1 . 28 1.46 1.38 1 .06JEWEL 1 . 77 1 . 87 2 . 09 2 . 05 2 . 65

Change in ais (g/lOOg

during storage fresh wt)

Months Curing 1 2 3 4BEAUREGARD 1.84 2 . 60 3 .57 4 . 07 4 . 82CENTENNIAL 1 . 74 3 .81 3 .41 3 . 85 4 . 51HERNANDEZ 3 . 64 4 .01 4 .14 5 . 00 5.41JEWEL 2 . 68 3 . 14 3.51 4 . 02 5 .60

Dry matter change plus total {g/100g fresh wt

sugar change)

Months Curing 1 2 3 4BEAUREGARD 1. 47 1. 92 2 .78 3 . 66 4 .01CENTENNIAL 1.36 3 . 63 3 . 28 3 . 18 3 .81HERNANDEZ 1.74 2 . 47 2 . 70 3 . 74 3 .40JEWEL 3 .00 3 . 50 3 . 94 4 . 44 6 .28

Change in dry matter during {g/100g fresh wt)

storage

Months Curing 1 2 3 4BEAUREGARD 0 .15 0 . 93 1 . 76 2 . 56 2 . 64CENTENNIAL -0.43 1 . 78 1 . 10 1 .45 1 . 75HERNANDEZ 0 . 71 1 .19 1 . 24 2 .36 2 .35JEWEL 1 . 23 1. 64 1 .85 2 .39 3 .63

1 Means of four readings.

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Figure 33. Dry matter contents in four sweetpotato cultivars at harvest (H), aftercuring (C), and during four months of storage.

145change with the summation of dry matter change and total sugar change, the difference became much smaller (Table 20). Therefore, the decrease in AIS is not only due to conversion of starch into sugars, but also due to respiration, which converts starch into CO, and H,0.

AIS contents after processing follow similar trends to AIS contents in raw roots. For all four cultivars, as length of storage increased, AIS content decreased (Figure 34 and Table 21). The largest decrease occurred in the first month of storage. 'Centennial' had the highest AIS contents,'Beauregard' and 'Hernandez' the lowest. During processing of puree, AIS decreased significantly from 40% to 50% (Table16). Compared to the ratios of maltose to AIS in raw roots, these values were slightly higher. This suggested that the decrease of AIS during processing was mostly due to conversion of AIS into maltose, with only a very small amount of short chain carbohydrates, which can be dissolved in the resulting alcohol solution.Effects of Storage on Viscosity of Sweetpotato Puree

For all cultivars, as length of storage increased, viscosity decreased (Figure 35 and Table 22) . The most significant change occurred after curing. During storage, viscosity of puree decreased at a much slower pace. For 'Centennial', viscosity appeared to increase after two months of storage. Among four cultivars, 'Centennial' had the highest viscosity at harvest, 'Jewel' had a much higher

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Figure 34. Alcohol-insoluble solids (AIS) content of processed sweetpotato puree forfour sweetpotato cultivars at harvest (H), after curing (C), and during four months ostorage.

147

Table 21. Effect of storage time on AIS of sweetpotato puree processed from four cultivars.

AIS (g/100 g fresh wt.)

Storagetime(month)


AT HARVEST 11.09 a1 15.63 a 10.64 a 15.07 aAFTER CURING 9.11 b 13 .76 ab 8.68 b 12 . 00 b1 MONTH 8.19 be 12 .31 b 8.08 b 11.25 b2 MONTHS 8.24 be 11. 75 b 8.66b 11.50 be3 MONTHS 7.92 be 12 . 56 b 7.98b 11.42 be4 MONTHS 7.52 c 12 .19 b 7.57 b 10.41 c


8.0BEAUREGARD

000s10w

CENTENNIAL

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0.0

H C 21 3


Figure 35. Viscosity of sweetpotato puree processed from four sweetpotato cultivars atharvest (H), after curing (C), and during four months of storage.

148

149

Table 22. Effect of storage time on viscosity ofsweetpotato puree processed from four cultivars.

Viscosity (CPSxlOOO)


AT HARVEST 3 .08 a1 7.27 a 3 . 05 a 5 . 71 aAFTER CURING 2 . 24 b 4 . 50 b 2 .28 b 2 . 87 b1 MONTH 1.98 b 3 . 51 b 1. 91 be 2 . 32 be2 MONTHS 2 . 06 b 2 . 77 b 1. 99 be 2 . 14 be3 MONTHS 1 . 92 b 3 . 86 b 1 . 92 be 2 .01 be4 MONTHS 1 . 55 b 3 . 99 b 1 . 47 c 1 .50 c


150viscosity than 'Beauregard' and 'Hernandez' at harvest, but it decreased to nearly the same value after four months of storage.

Since sweetpotato puree processing is actually an enzymatic hydrolysis of starch, the final viscosity of puree can be affected by substrate concentrations and enzyme activities. In another words, the viscosity of sweetpotato puree will depend on AIS content and a- and S-amylase activities in raw sweetpotato roots. From previous results in this study, no significant changes in a- and S-amylase activity occurred within cultivars during storage; only AIS decreased as the length of storage increased. It is not difficult to conclude that AIS content in raw roots is the most important factor that contributes to inconsistency of sweetpotato puree during four months of storage. However, it is difficult to demonstrate the results obtained among different cultivars. There were significant differences in amylase activities among cultivars. Since previous studies {Deobald et al. 1969; Walter et al. 1975) found that endogenous fi-amylase levels did not directly affect the amount of maltose produced during baking (no explanation was given for this phenomenon), inconsistency in sweetpotato products was thought mainly due to variable a-amylase levels. Therefore, it was expected that higher a-amylase activities would lower viscosity in sweetpotato puree, if a-amylases played a major role in determining the viscosity of puree. In

151this study, 'Centennial' had a much higher (a-amylase activity than 'Jewel', hence the viscosity should be lower, and maltose formed should be higher than in 'Jewel'. However, this is not what was found in this study; on the contrary, the viscosity of puree in 'Jewel' was lower and decreased more rapidly during four months of storage period than that of 'Centennial'.

Another unexpected result was also reported by Walter and Hoover (1984). It was found that starch conversion rate decreased in cooked strips of the 'Jewel' cultivar as the length of storage increased. Starch conversion rate andmaltose formation exhibited an apparent dependence on starchcontent not amylase activities. It was suggested that this effect may be due to a rapid temperature increase (steam at 160QC) in the strips inactivating enzymes before extensive starch conversion could occur. However, although a much lower processing temperature (80°C) was used in this study, results still show that maltose formation depended on AIS content, not amylase activities. One possible explanation is that a- amylase is heat labile with low activity as describedpreviously in this study, with most activity lost in just 30 seconds at 75°C. Its role in starch conversion could beinsignificant. As to the higher heat stability of S-amylase, it plays a major role in converting starch into maltose. Its levels do not affect the maltose produced probably because of following reason. It was reported that (Hassid and McCready,

1521943) when potato starch was treated with an excess amount of fi-amylase, the amylose fraction can be almost completely hydrolyzed, but the amylopectin can be hydrolyzed only to the extent of 54%. It Is well known that sweetpotatoes contain abundant fi-amylase, which could represent an excess amount of enzyme for the enzymatic hydrolysis in puree processing. In this case, most starch will be very rapidly hydrolyzed to maltose and the reaction will stop when only 6-limit dextrin is left, no matter how much fi-amylase is added. Therefore, for starch with the same amylopectin content, the maltose conversion rate should be the same and is not related to starch contents. The amount of maltose produced will depend on starch content. In chemistry, this type of reaction is known as a first-order reaction in which the reaction rate is directly proportional to the concentration of a single reactant, not the catalyst. The nearly consistent maltose conversion rates (Table 16) also suggest the saturation of fi- amylase in sweetpotatoes. From this point of view, it can be easily seen why endogenous fi-amylase levels did not directly affect the amount of maltose produced during heat treatment.

In addition, the relationships between viscosity versus AIS, and viscosity versus amylase activity were analyzed by SAS linear regression program. Apparently, AIS contents in raw roots are related to viscosity of puree (Table 23). The average correlation coefficient (R) of four cultivars is 0.82 (PsO.01). However, AIS contents after processing were more

153

Table 23. Relationships between viscosity of sweetpotato puree and AIS before processing (BP), AIS after processing (AP), a-amylase activity, and fi-amylase activity.

storage Correlation coefficients(r)1

r-Square(r1)Significance1

a-AMYLASE -0.21 0.04 *fl-AMYLASE -0.38 0.14 *AIS (BP) 0.82 0.67 **AIS (AP) 0.92 0. 85 **

1 Correlation coefficient r is average value of four cultivars.1 *, **, represent significance at P £ 0.05 and P £ 0.01.

154highly related with viscosity (R*0.92) . Only a very weak linear relationship existed between viscosity and amylase activities. Therefore, statistical results also suggest viscosity of puree is highly dependent on AIS content, with little dependence on a- and fi-amylase activity.Suggestions for Improving Consistency of Sweetpotato Puree.

Based on the results obtained in this study, inconsistent puree is caused by an AIS decrease in sweetpotato during storage; both a- and S-amylases have no significant effect on inconsistency of puree. Therefore, one basic requirement for producing a consistent product is that the AIS content be uniform. Generally, we can control the AIS by enzymatic treatment to reduce AIS or add starch back to increase AIS. For example, we assume that 1000 kg of 'Jewel' sweetpotato is going to be used for puree processing, and the optimum viscosity for the puree is 2 (CPSxiOOO) , from the data obtained in this study (Table 24) , which corresponded to AIS of 20.65% (g/fresh wt). However, at harvest, AIS was as high as 24.67%, the difference is about 4% (g/lOOg fresh wt. ) . Hence we can convert 4 g AIS in every lOOg fresh material into simple sugars by enzymatic treatment to decrease AIS to 20.65%. By calculation (4V/24.67% x 100% = 16%) , thus about 16% of sweetpotatoes, or 160 kg must be converted to simple sugar. Separating the sweetpotatoes into two portions: one portion with 840 kg will be processed by normal procedure, another portion will be subjected to

Table 24. Alcohol insoluble solids (AIS) contents in raw sweetpotatoes vs viscosity (VIS) of processed puree.1

Storage Cultivartime -------------------------------------------------------------(month) Beauregard Centennial Hernandez Jewel

AIS VIS AIS VIS AIS VIS AIS VISAT HARVEST 19.47 3 .08 26.20 7.27 20.23 3 .05 24.67 5.71

AFTER CURING 17.64 2 .24 24 .46 4.50 16 .59 2.28 21. 99 2.87

1 MONTH 16.87 1.98 22.39 3.51 16.22 1.91 21. 54 2.322 MONTH 15.91 2 .06 22.79 2.77 16 .09 1. 99 21.16 2.143 MONTH 15 .40 1.92 22.35 3.86 15.23 1. 92 20.65 2.014 MONTH 14 .65 1.55 21.69 3. 99 14 .82 1.47 19.07 1.50

1 This table was summarized from Table 19 and Table 22 to provide reference for establishing optimal processing procedure.

155

156complete hydrolysis by saccharifying enzymes, e.g. a-amylase and pullulanase (debranching enzyme) . At the end of processing the two portions will be combined and a productwith viscosity 2 CPSxlOOO could be obtained. As the length ofstorage increase, AIS could decrease below 20.65%, e.g.,19.07% after four month of storage. Adding AIS back would then be needed instead of enzymatic treatment. AIS can be prepared following normal sweetpotato starch production procedure, except that there would be no need to remove fiber or other materials by filtration. The amount of AIS added can be calculated as follows: (20.65% - 19.07%)/(1-20.65%) x 1000 kg = 19.91 kg. This amount of AIS must be mixed very wellwith 1000 kg sweetpotatoes before cooking to achievesatisfactory results. Also starch can be added back instead of AIS. But the relationship between viscosity and starch content in sweetpotatoes should be established.

CHAPTER V SUMMARY AND CONCLUSIONS

A critical problem associated with the production of sweetpotato puree is inconsistent final products since sweetpotato roots undergo internal changes during storage. It was generally believed that the nature of this change was due to the increase of amylase activity in sweetpotatoes. However, puree processing is actually an enzymatic hydrolysis procedure, which could be affected not only by enzyme levels but also by substrate concentrations. Therefore, to understand the effects of both factors on inconsistency of puree, the changes in amylase activity and carbohydrates of sweetpotatoes during storage were investigated.

Since sweetpotatoes contain both or- and S-amylase, there is a need to eliminate interference between these two enzymes when measuring activity. A method for the specific determination of a-amylase (using BPNPG7 as substrate) and S- amylase (using PNPG5 as substrate) were adapted for amylase assays in sweetpotatoes. Both methods have met the two main criteria in the development of assays with crude extracts:(1) that activity is proportional to the quantity of extract used, and (2) that activity is linear over the assay period chosen. In addition, both methods correlate well (r>0.99) with other well-established procedures for the assay of a-

157

158amylase and S-amylase, but have the major advantages of simplicity, rapidity, and specificity. In the examination of specificity, the BPNPG7 assay method showed that BPNPG7 responds to a-amylase only and is not affected by S-amylase. A further advantage of BPNPG7 assay procedure is its extreme sensitivity; ten times more sensitive than amylose azure, twenty times more sensitive than starch azure. It is especially useful for measuring a-amylase activity in sweetpotatoes. The PNPG5 method is not completely specific for S-amylase, although it has a much higher specificity than the Bernfeld method. However, it is still possible to measure activity accurately by using the established equation. In sweetpotatoes, because the a-amylase level is very low, its effect on PNPG5 can be negligible.

All a-amylases in the four cultivars tested had similar Km values for hydrolysis of BPNPG7, ranging from 0.55 to 0.65 mM. The S-amylase in 'Centennial' had significantly higher Km value for hydrolysis of PNPG5 at 1.73 mM. The other three cultivars had similar Km values, which ranged from 0.82 to 0.91 mM.

In general, S-amylase in sweetpotatoes has a higher thermal stability than a-amylase. Heat inactivation patterns at 75°C show that a-amylase lost most of its activity in just 30 seconds of heating, then slowly declined to 13V of the original value after 5 min of heating. For S-amylase, about 60% of activity still remained after 10 min of heating. Low

159temperature storage also showed S-amylase is more stable than a-amylase. During four weeks of storage at room temperature, and 4°C, no activity loss was observed in S-amylase. Among four cultivars, a-amylase in 'Jewel' is the least heat stable. S-amylase in 'Centennial' is the most heat stable. Eight times dilution of sweetpotato juice had a significant effect on S-amylase stability but little effect on a-amylase stability.

When a high concentration of amylase was incubated with gelatinized sweetpotato starch, the combined action of a- amylase and E-amylase was more efficient than the action of either alone, based on same activity level. However, no significant difference was observed at normal assay concentrations. A possible mechanism was suggested.

The a- and S-amylase activities showed significant differences among cultivars except between 'Beauregard' and 'Hernandez'. 'Centennial' had the highest a- and S-amylase activities, 'Jewel' had the lowest a-amylase activity, 'Beauregard' and 'Hernandez' had the lowest S-amylase activity. During four months of storage, for all cultivars, both a- and S-amylase activities remained unchanged (P<0.05).

For all four cultivars, storage generally increased the sugar contents in sweetpotatoes, with the greatest increase occurring during the curing period. After curing, sugar content increased at a much slower pace. On the other hand, the alcohol insoluble solids (AIS) contents decreased as

160length of storage increased. Most of the decrease occurred in the first month of storage. The decrease of AIS is due partly to starch being converted to reducing sugars and sucrose. Respiration that converts starch into CO, and H,0 is another important reason. During puree processing, a large amount of AIS was converted to maltose, the maltose conversion rate was about 40% and was consistent for all cultivars. The amount of maltose produced decreased as storage time increased. These results suggested the "saturation" of S-amylase in sweetpotatoes and explained why S-amylase levels do not directly affect the amount of maltose produced during heat treatment.

The effect of root storage on viscosity of sweetpotato puree was investigated. In general, viscosity change followed a pattern similar to that of AIS during four months of storage.

The relationships between viscosity and AIS, and viscosity and amylases activities were analyzed by SAS linear regression program. Statistical results further suggested that viscosity of puree had a strong dependence on AIS content, but little dependence on a- and S-amylase activities.

Apparently, the viscosity in sweetpotato puree is AIS dependent, due to the following considerations: (1) Nosignificant amylase activity change occurred during storage;(2) Change in AIS pattern during storage almost mirrored

161viscosity change pattern; (3) S-amylase levels did not affect maltose produced during heat treatment due to "saturation1'; (4) a-Amylase was heat labile with low activity, thus its role in puree processing could be insignificant; (5) Statistical results showed high correlation between viscosity and AIS, and low correlation between viscosity and amylase activities; (6) If viscosity were a-amylase dependent, 'Jewel', which had a much lower a-amylase activity than 'Centennial', should have had a higher viscosity.

In conclusion, two amylase assay procedures, PNPG5 and BPNPG7, have the major advantages of simplicity, rapidity, high sensitivity, and specificity for determination of sweetpotato £-amylase and a-amylase. These assays should prove valuable for laboratory use and also for production control during processing.

During storage of sweetpotatoes, the a- and £-amylase activities had no significant changes and produced no significant effect on consistency of sweetpotato puree. The inconsistent products in sweetpotato puree processing are most likely due to AIS changes in sweetpotatoes during storage. The decrease of AIS is partially due to respiration that converts starch into CO, and H,0. Therefore, the basic requirement for reliably producing a consistent puree is that the AIS content be uniform. It is possible to control the AIS by enzymatic treatment to reduce AIS or adding AIS back to increase AIS. The results obtained from this research could

162be useful to aid in establishing optimal processing procedure for improving inconsistency in sweetpotato processing.

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VITA

The author was born in Fujian Province, China on October 30, 1963. His primary and secondary education was acquired in Changsha, Hunan.

After graduation from The First Middle School of Changsha, he entered Hunan Agricultural University in 1980 and was granted a Bachelor of Science in Food Science in 1984 .

After graduation, he entered graduate school at Hunan Agricultural University and was granted a Master of Science in Biochemistry in 1987.

In 1987, he accepted a product development scientist position in the China Tea Import and Export Corporation, Department of R&D.

In 1991, he accepted a research assistant position in the Department of Horticulture at Louisiana State University to work toward the Doctor of Philosophy in Horticulture with specialization in food processing and postharvest.

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DOCTORAL EXAMINATION AND DISSERTATION REPORT

C a n d id a te ! Xiangyong Liu

M a jo r P i e l d t Horticulture

T i t l e o f O i a a e r t a t i a o i Changes of Amylases and Carbohydrates InSweetpotatoes during Storage and Their Effects on Viscosity of Sweetpotato Puree

M a jo r P r o f e a a o r a n d C h airm anPaul VL Wilson

EXAMINING COMMITTEE:

U MSamuel Godber

Robert M. Grodner

Don R. Labonte

D a t e o f K x a m in a t lo m

June 23, 1995

changes of amylases and carbohydrates in sweetpotatoes

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